M受体亚型、血管内皮生长因子、内皮抑素在人前列腺组织中的表达及意义
摘要
研究背景与目的：
前列腺是成年男性的重要器官，具有分泌前列腺液、协助射精、排尿等多种重要功能。前列腺生理功能的紊乱及病理变化可以导致许多疾病的发生，严重影响男性的身心健康。良性前列腺增生(BPH)是我国男性发病率最高的良性肿瘤，60岁以上老年人有50％发生良性前列腺增生症也是最常见的老年病致死原因，前列腺癌(PCa)一直是西方国家男性最常见的恶性肿瘤。在我国尽管前列腺癌的发病率相对较低，但近几年也有逐年上升的趋势。对前列腺癌进行早期发现和有效的治疗已使总体死亡率有了明显的降低。然而，晚期前列腺癌的治疗仍然是一个难题。慢性前列腺炎由于其不适症状重、治疗效果不满意，影响着患者的正常生活，因而也一直困扰着国内外泌尿外科医生。前列腺疾病的危害如此之大足以引起我们的重视，然而前列腺这些疾病的发病机理很多还不清楚，治疗效果还很不满意，还需要进一步深入研究。
前列腺的生长与功能受多种神经系统的调节，包括胆碱能神经与去甲肾上腺素能神经，近年来对前列腺的组织结构与功能的研究已经越来越深入和广泛，特别是对调节前列腺功能的植物神经系统的研究显示前列腺疾病发生、发展与神经系统的调节关系密切。在良性前列腺增生症的治疗上α受体阻滞剂已经取得了巨大的成功，雄激素阻断也已经成功地用于前列腺癌的内分泌治疗。然而对于胆碱能M受体在前列腺生理功能与疾病发生发展中的作用还不是很清楚，也还没有这方面的药物应用于前列腺疾病的治疗。
认识胆碱能M受体在前列腺中的作用，并把胆碱能药物用于前列腺疾病的治疗，首要的问题是弄清楚M受体在前列腺组织的分布特征，目前在良性前列腺增生组织及前列腺肿瘤细胞系已有这方面的研究，但是在正常前列腺各区带的分布规律目前还很少有研究报道，可查的资料也是本课题组2006年关于3种M受体亚型在正常前列腺表达规律的研究报道。
最近几年，关于前列腺癌发生和进展中发生的分子生物学改变方面的知识增加迅速，对肿瘤分子生物学更好的理解对于建立新的对策来延缓或抑制肿瘤的进展具有重要的意义。
研究表明在许多肿瘤细胞都有胆碱能受体表达，并且表达强度高于正常。对乳腺癌及其他肿瘤细胞系的研究发现，M_3受体与肿瘤的发生、分级密切相关，胆碱能受体的活动通过不同的信号途径可以刺激细胞的增殖。既往的研究发现M_1、M_2、M_3能刺激细胞的生长与分化，而M_4的这种功能则不明显。并且起调节作用的主要是M_3受体。这说明M_3受体表达的变化在肿瘤发生中具有重要的作用，了解M_3受体在正常前列腺、良性前列腺增生、前列腺癌组织中的表达变化规律对于探讨其在前列腺肿瘤发生、发展中的作用及其分子生物学机制具有重要的意义。
在肿瘤的发生、发展过程中，肿瘤细胞的增殖与新生血管的形成是重要的环节，在乳腺癌的LMM3细胞系的研究发现，其主要受体亚型为M_3，且表达量是LM3细胞的40倍。胆碱能受体激动剂CARB能够刺激LMM3细胞的生长与新生血管生成，选择性的M_3受体拮抗剂pf-HHSiD能抑制这种作用，并且试验表明CARB能刺激VEGF-A的表达，选择性的M_3受体拮抗剂pf-HHSiD也能抑制这种作用。这说明M_3受体在LMM3细胞的生长与血管生成中起着十分重要的作用。目前关于M_3受体与良性前列腺增生、前列腺癌发生发展的关系方面的研究还很少，并且已有的研究也是在没有严格意义上的正常对照的基础上完成的。
血管生成受到正负两方面因子的调控：血管生成刺激因子和血管生成抑制因子。血管内皮生长因子(vascular endothelial growth factor，VEGF)和内皮抑素(Endostatin ES)分别是目前已知的最强的血管生成促进因子和抑制因子，联合检测二者在肿瘤组织中的表达有助于更加深入地了解促血管生成因素和抑制血管生成因素在血管生成和肿瘤浸润转移中的作用。
血管内皮生长因子是肿瘤血管新生的重要作用因子。在绝大多数恶性肿瘤中均呈现VEGF及其受体的高表达，以旁／自分泌作用机制，促进内皮细胞分裂、增殖、诱导血管新生，并可直接作用于肿瘤细胞，刺激肿瘤细胞生长。在前列腺内VEGF可由正常导管内皮细胞、腺癌细胞和浸润的淋巴细胞合成。
VEGF的表达随着人类前列腺癌细胞的转移特性的增加而增加，而且恶性肿瘤细胞合成、分泌的VEGF刺激血管生成的效率更高。VEGF受体在前列腺肿瘤血管内皮细胞和肿瘤细胞上均有表达，这说明VEGF除了以旁分泌刺激血管内皮细胞增殖外，还能通过自分泌调节肿瘤细胞增殖。
Endostatin是从血管内皮瘤培养液中分离出的18型胶原蛋白C端氨基酸片段。它可特异性地抑制内皮细胞增殖、迁移和肿瘤血管生成，诱导细胞凋亡，使肿瘤原发灶及转移灶处于休眠状态，停止生长。
文献报道肿瘤内微血管密度(microvessel density MVD)反映了肿瘤间质血管生成的强度，随着MVD的增高，肿瘤的复发呈上升趋势。目前MVD已经成为评价肿瘤组织内新生血管形成程度的重要指标。
研究前列腺组织中VEGF与内皮抑素、M_3受体三者在前列腺正常组织与肿瘤组织中的表达关系对于研究M_3受体在前列腺肿瘤发生发展中的作用，对于深入研究前列腺肿瘤的发生与发展机制以及对于晚期肿瘤治疗靶点的选择均具有重要的意义，此研究在人体前列腺肿瘤还未查见相关报道。
材料与方法：
本研究分两部分进行，第一部分，首先应用实时荧光定量RT-PCR方法对M受体及其各亚型在正常前列腺各区带的mRNA表达进行研究，探讨各亚型在各区带的基因表达差异，然后进一步对在前列腺组织表达较高并与肿瘤发生密切相关的三种受体亚型(M_1、M_2、M_3)通过Western blot方法检测其在正常前列腺、良性前列腺增生、前列腺癌组织中的表达差异，获得各亚型在不同前列腺组织中的蛋白表达特征，分析不同亚型在前列腺正常功能发挥与疾病发生、发展中的作用。第二部分，先通过RT-PCR方法研究M_3、VEGF、ES在正常前列腺、良性前列腺增生、前列腺癌三种组织的基因表达，再应用免疫组化及Western blot两种方法并对比CD34的表达综合对比VEGF与内皮抑素在正常前列腺、良性前列腺增生、前列腺癌三种组织中的蛋白表达差异，分析这两种物质的表达在前列腺肿瘤发生、发展中的作用及与前列腺癌临床分期、gleason评分的关系：最后通过与M_3受体在正常前列腺与良性前列腺增生、前列腺癌中的表达进行对比研究，进一步探讨M_3受体在前列腺肿瘤发生、发展中的作用。这对于以后前列腺疾病治疗药物靶点的开发具有重要的意义。
取材：
RT-PCR及Western blot检测采用新鲜标本，其中正常前列腺组织采用健康成人新鲜尸体前列腺标本，良性前列腺增生、前列腺癌标本为采用前列腺切除手术或穿刺活检取得的标本(取自山东大学第二医院及渮泽市立医院2003-2006年的住院病人)，所有标本取出后均放于液氮中，转-80℃冰箱保存，实验前取出复温。检测M受体亚型在前列腺各区带的表达所用正常前列腺标本7例，检测M_3受体、VEGF、内皮抑素基因与蛋白表达所用标本分别为：正常前列腺36例、良性前列腺增生36例、前列腺癌8例。
免疫组化标本采用石蜡包埋标本，正常前列腺组织来自健康、新鲜尸体前列腺标本，良性前列腺增生、前列腺癌标本为采用前列腺切除手术或穿刺活检取得的标本(取自山东大学第二医院2002-2006年的住院病人)，标本经10％福尔马林浸泡，石腊包埋。其中正常前列腺36例，良性前列腺增生36例，前列腺癌36例。
操作在超净台进行，取新鲜标本时，每取一个部位均更换手术刀片，正常前列腺组织每个标本取相邻3块，分别做RT-PCR与Western blot及做石腊块用于免疫组化。
实验方法：
一、实时荧光定量RT-PCR方法检测正常成人前列腺各区带M受体各亚型(M_1～M_5)基因表达
实验所用物品的去RNase处理，将组织低温下研碎，组织提取总mRNA，合成cDNA，进行PCR扩增反应，荧光定量RT-PCR反应条件：95℃10s，95℃5s，60℃45s，40个循环；得到每个部位、每个受体的Ct值，用△Ct法转换为其初始相对浓度。其换算公式为：△Ct=Ct_(样本)-Ct_(β-actin)，平均相对含量=2~(-△Ct)。
统计学处理
应用SAS9.0软件进行统计学处理，统计方法为配伍组设计资料的方差分析。并应用q检验进行组间两两比较，P＜0.05认为差异有统计学意义。
二、RT-PCR方法检测M_3受体、VEGF、内皮抑素在正常前列腺、良性前列腺增生、前列腺癌组织中的基因表达
实验所用物品的去RNase处理，组织低温下研碎，提取总RNA，进行逆转录(RT)反应合成cDNA。聚合酶链反应(PCR)。定量分析：将凝胶电泳图像输入美国Kodak凝胶分析系统，应用1 D Image Analysis Software进行表达强度分析，按下公式计算相对系数。
相对系数=细胞因子表达强度／β-actin表达强度
统计学处理
应用SAS9.0软件进行统计学处理，统计方法为随机设计资料的方差分析。应用q检验进行组间两两比较，P＜0.05认为差异有统计学意义。M_3与VEGF采用pearson相关性分析。
三、免疫组化法检测VEGF、内皮抑素、CD34在正常前列腺、良性前列腺增生、前列腺癌组织中的蛋白表达
首先将蜡块连续切片4片，片厚4μm。取胎盘组织以PBS代替一抗做阴性对照，把每组标本的3片分别做VEGF、内皮抑素及CD34免疫组化，另一切片做HE染色。根据HE染色，对每个样本进行病理学审查(由病理科医生协助完成)，排除病变样本。结果判定以染色阳性物质呈棕黄色。M_3、VEGF、内皮抑素染色结果判定：根据阳性细胞显色深浅及阳性细胞百分率分级：①显色深浅：不显色0分；浅黄色1分；棕黄色2分；深褐色3分。②阳性细胞百分率：无阳性细胞显色0分；阳性细胞显色＜25％为1分；25％～50％为2分；阳性细胞显色＞50％为3分。二者得分相加：0分—阴性(-)；1～2分—弱阳性(+)；3～4分—阳性(++)；5～6分—强阳性(+++)。结果判断在双盲下进行，每张切片由两位病理医师分别阅片记数。CD34染色阳性的单个内皮细胞或一簇内皮细胞作为一个微血管，在200×视野下计数3个血管密度最高区域(热点)的微血管数目，其平均值即为肿瘤的微血管密度(MVD)。
统计学处理
应用SAS9.0软件进行统计学处理，统计方法为随机设计资料的方差分析与x~2检验，P＜0.05认为差异有统计学意义。M_3与VEGF采用pearson相关性分析。
四、Western blot方法检测三种M受体亚型(M_1、M_2、M_3)、VEGF、内皮抑素的蛋白表达
取新鲜组织块，称量后提取蛋白(M受体提取膜蛋白、VEGF、内皮抑素提取胞浆蛋白)、加上养液、煮沸、在Bio-Rad垂直电泳仪中灌注质量分数10％分离胶和质量分数7.5％浓缩胶，加入不同组样本，稳压120V电泳90min，然后在转膜仪上将凝胶上的蛋白转移到PVDF膜上，稳压100V转移60min，取出膜后用7％脱脂牛奶封闭60min，加入一抗，4℃过夜，次日加入抗兔二抗孵育2h，DAB显色，扫描图像，输入美国Kodak图像分析系统，应用1 D Image Analysis Software进行表达强度分析，按下公式计算相对系数。
相对系数=细胞因子表达强度／β-actin表达强度
统计学处理
应用SAS9.0软件进行统计学处理，统计方法为随机设计资料的方差分析。应用q检验进行组间两两比较，P＜0.05认为差异有统计学意义。VEGF与ES采用pearson相关性分析。
结果：
第一部分
1、荧光定量RT-PCR方法检测结果显示五种M受体亚型在正常成人前列腺均有不同程度的基因表达，各个区带各受体的表达存在差异。以M_2、M_3、M_1受体的表达为主。
在前纤维肌区M_2的表达明显高于其他受体(P＜0.05)，约是M_3表达量的3倍；M_3、M_1、M_5、M_4的表达差异无统计学意义(P＞0.05)。
在移行带M_2的表达明显高于其他受体(P＜0.0001)，约是M_3表达量的三倍。M_3的表达明显高于M_4(P＜0.05)。
在中央带M_2的表达最高，但只与M_4之间比较差异有统计学意义(P＜0.05)；M_2、M_1、M_3、M_5四者间差异无统计学意义(P＞0.05)。在外周带各受体亚型的表达差异均无统计学意义(P＞0.1)。
2、Western blot检测显示M_1、M_2、M_3在正常前列腺、良性前列腺增生、前列腺癌组织中的蛋白表达存在差异。
在正常前列腺M_2的表达高于M_3，更高于M_1，三者比较差异均具有统计学意义(P＜0.01)。
在良性前列腺增生与前列腺癌组织M_2、M_3受体的表达高于M_1，差异具有统计学意义(P＜0.01)。M_2、M_3受体之间的表达差异无统计学意义(P＞0.05)。在正常前列腺与病变前列腺组织中表达差异具有统计学意义。
M_1受体在正常前列腺、良性前列腺增生、前列腺癌三种组织的表达差异无统计学意义(P＞0.05)。
M_2受体在正常前列腺、良性前列腺增生、前列腺癌三种组织的表达差异无统计学意义(P＞0.05)。
M_3受体在前列腺癌组织的表达高于在良性前列腺增生组织的表达，正常前列腺组织的表达最低，差异均具有统计学意义(P＜0.01)。
第二部分
1、RT-PCR结果显示M_3受体、VEGF、内皮抑素在正常前列腺、良性前列腺增生、前列腺癌组织的基因表达存在差异
M_3受体在不同前列腺组织的基因表达存在差异，表达顺序为：前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义(P＜0.01)。
VEGF在不同前列腺组织的表达不同，表达顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义(P＜0.01)。
内皮抑素在不同前列腺组织的表达不同，其均值顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均不具有统计学意义(P＞0.05)。
M_3受体的表达与VEGF的表达具有正相关关系(r=0.4999 P＜0.001)。
2、免疫组化结果显示在正常前列腺、良性前列腺增生、前列腺癌组织VEGF、内皮抑素的蛋白表达存在差异
CD34在不同组织的表达强度顺序为：前列腺癌＞良性前列腺增生＞正常前列腺(P＜0.05)。
MVD与前列腺癌临床分期的关系比较，因为样本含量少，合并临床Ⅰ、Ⅱ期与临床Ⅲ、Ⅳ期相比，P＞0.05，差异无统计学意义。
MVD与前列腺癌Gleason评分的关系比较，评分≥8分的与评分＜8份的比较，P＞0.05，差异无统计学意义。
VEGF在不同组织的表达强度顺序为：前列腺癌＞良性前列腺增生＞正常前列腺，差异均具有统计学意义(P＜0.01)。
ES在不同组织的表达强度顺序为：前列腺癌＞良性前列腺增生＞正常前列腺，差异均具有统计学意义(P＜0.01)。
VEGF与前列腺癌临床分期的关系比较，因为样本例数含量少，合并临床Ⅰ、Ⅱ期与临床Ⅲ、Ⅳ期相比，差异有统计学意义(P＜0.01)。
VEGF与前列腺癌Gleason评分的关系比较，评分≥8分的与评分＜8分的比较，差异有统计学意义(P＜0.05)。
ES与前列腺癌临床分期的关系比较，因为样本含量少，合并临床Ⅰ、Ⅱ期与临床Ⅲ、Ⅳ期相比，差异具有统计学意义(P＜0.01)。
ES与前列腺癌Gleason评分的关系比较，评分≥8分的与评分＜8分的比较，差异具有统计学意义。(P＜0.05)
3、Western blot检测结果显示VEGF、内皮抑素的蛋白表达存在差异
VEGF在不同前列腺组织的表达不同，表达顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义。(P＜0.05)
内皮抑素在不同前列腺组织的表达不同，其均值顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义(P＜0.05)。
VEGF的表达与内皮抑素的表达具有正相相关(r=0.5582 P＜0.001)。
结论：
1、前列腺各个区带均有不同程度的5种M受体亚型的基因表达，主要以M_2、M_3、M_1为主。
2、M_2、M_3、M_1受体在正常前列腺的蛋白表达与基因表达具有相同的趋势，M_3受体在前列腺肿瘤组织的蛋白表达高于在正常前列腺的表达，与前列腺肿瘤的发生、发展关系密切。
3、VEGF、ES与前列腺肿瘤的发生及前列腺肿瘤的临床分期、gleason评分有密切关系。
4、M_3、VEGF、ES在前列腺组织的表达具有正相直线相关关系。
5、M_3、VEGF、ES、CD34可进一步研究作为前列腺肿瘤诊断指标及治疗新靶点的选择。
Research background and objects:
Prostate which is an important organ of male adult has many important functions, such as prostatic fluid secretion, ejaculation assisting, urination and so on. The physiological and functional disorder of the prostate and its pathological changes can cause many diseases, even affect the physical and mental health of men. Benign prostatic hyperplasia is,of the highest incidence disease among tumors of men in our country. Prostate cancer is one of the most common malignant tumors for men in western country. Although the incidence of prostate cancer is low in our country, it has the tendency to increase in recent years. Chronic prostatitis affects patients' life because of severe symptoms and unsatisfied treatment, so that it continuously harasses urologists all over the world. Prostatic diseases' disadvantages are serious enough to call our attention, but the mechanism of these diseases is still unknown and the treatment is not so satisfied.
Detective and aggressive treatment of early prostate cancer resulted in a significant decline of overall mortality rates. However, the treatment of advanced tumor stages is still a crucial problem. Knowledge on molecular alterations taking place in the development and progression of prostate cancer increased rapidly in the past few years, thanks also to the use of novel techniques that enabled to perform broad gene expression profiling of tumors. This better understanding of the molecular biology of prostate cancer led to the establishment of novel strategies to delay/inhibit tumor progression.
The growth and function of prostate is regulated by many nervous systems, including cholinergic and epinephrine nerves systems. In the past few years the construction and function of prostate have been deeply researched. The researches of autonomic nerve system's regulation for function of the prostate demonstrated that the regulation of nerve system was intimately related to the genesis and development of prostatic diseases. Great success has been obtained in the research of alpha receptor inhibitor used in the treatment of benign prostatic hyperplasia, and androgen-blocking has been successfully used in the endocrine treatment of prostate cancer. But cholinergic muscarinic receptor's functions in regulating the prostatic physiologic function, disease genesis and development is still unclear. And also there has been no such kind of drugs used in the treatment of prostatic diseases.
In order to better understand the roles of cholinergic muscarinic receptors in prostate and to apply cholinergic drugs in the treatment of prostatic diseases, we should make out the distribution characteristics of muscarinic receptor in normal human prostatic tissue. Up to now there are only researches about muscarinic receptor on benign hyperplastic prostate tissue and prostatic tumor cell lines, but the report of distribution regularity in different zones of normal human prostate is only fewer. The only report is done by our research team about the distribution regularity of the three kinds of muscarinic receptor subtypes in normal human prostate in 2006
The cholinergic receptor expression was prevalent in many tumors, in which mostly higher than in the normal tissue. The research about breast carcinoma and other tumors revealed that M_3 receptor was intimately related with the carcinogenesis and classification of tumor. The activity of cholinergic receptor could provoke the cell proliferate through different signal pathway. The previous research found out that M_1, M_2. M_3 receptors could stimulate cell growth and differentiation, but M_4 was not found having such funetions. Furthermore M_3 receptor is the main subtype to play the role of regulator, and be changed much in tumor generation. To understand the expression rule of M_3 receptor in healthy, hyperplastic, cancerous prostate tissue is very important for making clear the role of M_3 receptor and its molecular biologic mechanism in genesis and development of prostatic tumor.
The proliferation of tumor cells and the formation of new blood vessels were important steps in the process of the genesis and development of tumor. In the research of breast cancer LMM3 cell line, it was found that the main subtype was M3 and its expression quantity was 40 folds of LM3 cell. The cholinergic receptor agonist CARB could provoke the growth and neovascular genesis of LMM3 cell, but this function could be suppressed by the selective M3 receptor antagonist. It was also demonstrated that CARB could promote the expression of VEGF-A, which could be inhibited by selective M3 receptor antagonist pf-HHSiD. All these findings told us that M3 receptor played an important role in LMM3 cell growth and neovascular genesis. But the researches about M 3 receptor in prostatic hyperplasia and cancer was much fewer, and the fewer finished study was done based on not strict normal control.
Vascular endothelial growth factor(VEGF) is an important factor in neogenesis of tumor vessels. VEGF and its receptor are highly expressed in most malignant tumors, through para/autoendocrine reaction, promoted endothelial cell division, proliferation and induced vascular neogenesis, directly affecting tumor cell and provoking it to grow. In prostate, VEGF could be synthesized by normal duct endothelial cell, adenoma cell and infiltrative lymphocyte.
The stronger the metastatic characteristic of human prostatic cancer cell was, the higher VEGF expression would be, and neovascular genesis would be more effectively promoted by VEGF which was synthesized and secreted by malignant cell. The VEGF receptor expressed on both the vessel endothelium of prostatic tumor and the tumor cell showed that VEGF not only stimulated vascular endothelial proliferation by paraendocrine way but also regulated tumor cell proliferation by autoendocrine way.
Endostatin was XVIII collagen C-terminal amino segment which was isolated from culture solution of vascular endothelioma. It could specifically inhibit endothelial cell proliferation, metastasis and tumorous angiogenesis, and could induce cell apoptosis, so that it made the primary and metastatic site of tumor stay in dormant state and stop growing.
To study VEGF, endostatin and M_3 receptor's relation in normal and tumor prostate is very important for further understanding the mechanism of tumorigenesis,development, and selecting the target of treatment in late stage tumor. At present the research on this aspect about human prostatic tumor has not been reported.
For the above reason my research firstly studied the mRNA expression of muscarinic receptor subtypes in each zone of normal prostate and discussed the expression difference of each subtype among different zones. Then we detected the expression of those three kinds of receptors, among the normal, hyperplastic and cancerous prostate, using Western blot, which were highly expressed in the prostatic tissue and had intimate relationship with the tumor genesis. We gained the protein expression difference of each subtype in different zones of prostate tissue and analyzed the role of different subtype in normal human prostate,and in the genesis.developmeiit of diseases. At last using RT-PCR, immunohistochemistry, Westsern blot, we comparatively studied the expression of VEGF and endostatin in prostatic tissue at genetic and protein levels, together with the expression of M_3 receptor in normal, hyperplastic and cancerous prostate, and discussed their roles in genesis and development of prostatic tumor.
These researches were important for understanding M receptor, VEGF and endostatin's roles in prostate's function educing and disease generation, also it is more important for developing new drugs for prostatic diseases .
Material and method:
Materials:
The fresh normal prostates were obtained from healthy adult donators who were died of accident. The samples of hyperplastic, cancerous prostates were taken from the hospitalized patients in The Second Hospital of SDU from 2000 to 2006, and from prostatectomy or core biopsy. All the tissues were put in nitrogen liquid and kept in -80℃refrigerator for further using. Seven samples of normal prostate were used in detecting M receptor subtype in each zone of prostate. The specimens used in detecting M_3 receptor, VEGF, endostatin at genetic and protein levels included 36 normal prostates, 36 hyperplastic ones, and 8 cancerous ones.
The samples detected with immunohistochemistry were paraffin-embedded ones. The normal prostatic tissue was from fresh corpses of healthy adults. The hyperplastic, cancerous samples were from hospitalized patients of The Second Hospital of SDU from 2000 to 2006, taken with prostatectomy or care biopsy. The samples were soaked in 10% formalin and paraffin-embedded. There were 36 normal prostate, 36 hyperplastic ones and 36 cancerous ones. All the operations were taken on super clean bench. During taking fresh samples, the operating knife blade should be changed for different site. Every sample of normal prostate was taken out 3 pieces, respectively used in RT-PCR,western blot and made into paraffin-embedded block which would be used in immunohistochemistry.
Methods:
I .Detect muscarinic receptor subtypes'genetic expression in each zone of normal human prostate with real-time fluorescent quantitative RT-PCR
Process the experiment material with RNase. Levigate the tissue at low temperature. Extract the total mRNA. Synthesize cDNA. Carry out PCR amplification reaction. Real Time PCR reaction condition: 95℃10s, 95℃5s, 60℃45s, 40 cycles; Every receptor's threshold cycle(Ct) value of each site which we got was transformed into its initial relative concentration with△Ct way. The transformation formula:△Ct=Ct_(sample)-Ct_(β-actin). The mean relative quantity = 2~(-△Ct) . Apply SAS9.0 software in statistical treatment. The statistical method was variance analysis to pair-matched data. And q test was used to compare every two groups. The difference was considered as statistically significant when P<0.05.
II .Using RT-PCR detect M_3 receptor, VEGF, endostatin's expression in normal,hyperplastic, cancerous prostate at genetic level:
The experiment material was processed with RNase. Levigate the tissue at low tempreture. Extract the total RNA.The RNA was then reverse transcribed into cDNA. Input gel electrophoresis picture to the American Kodak gel analystic system.Use 1 D Image Analysis Software to analyse the expression intensity.Calculate the relative coefficient with the formula below.
The relative coefficient=cytokine expression intensity/β-actin expression intensity
III. Detect VEGF, endostatin, CD34's expression in normal, hyperplastic, and cancerous prostate with immunohistochemical method
Firstly we cut 4 serial sections with thickness 4 micrometres from the paraffin block. Fetal tissue with PBS instead of first Ab serves as negative control. The 3 slices of every group were respectively used in VEGF , endostatin and CD34's immunohistochemical detection. The other one was HE stained. Every sample was inspected by the pathologist. In the result evaluation the positive stained should be brown. M_3, VEGF, endostatin's staining result assessment: to classify them according to the extent of positive cell coloration and positive cell percentage: i according to the coloration:no color score 0;buff 1;buffy 2;sable 3. ii according to the positive cell percentage: no positive cell score 0; positive cell<25% 1; positive cell 25%-50% 2; positive cell>50% 3;Then add the two score : 0-negative(-);1-2-weakly positive(+); 3-4-positive(++);5 -6-strongly positive(+++).The result assessment was carried out with double blind method. Every slice was inspected and counted by two pathologists respectively. We considered one single endothelium or one cluster of endothelium, which was CD34 positive stained as one microvessel. We counted the microvessel number of 3 different visual field where the vessel density was the highest in 200X view field. The mean value was treated as tumor's microvessel density.
iv. Detect M_1, M_2, M_3, VEGF and endostatin's protein expression in normal, hyperplastic, cancerous prostate with western-blot method
Fresh tissue were taken and weighted.Then the protein(we extracted membrane protein for M receptor, extracted the cytoplasmic protein for VEGF and endostatin) was extracted and denatured.Different samples were added in Bio-Rad vertical electrophoresis apparatus which had been perfused with weight percentage 10% separating gel and weight percentage 7.5% stacking gel, and ran for 90 min at stable voltage 120 volt. Then buffer transferred the protein in the gel onto PVDF membrane with transferring membrane, at stable voltage 100 volt for 60 min. The membrane was taken out and blocked by 7% skimmed milk for 60 min. Then it was incubated with first Ab overnight at 4℃, washed thoroughly and with the second Ab for 2 hours. Stained the membrane with DAB and scanned the picture. The picture was inputted into the American Kodak Image Analystc System. Using 1 D Image Analysis Software analyzed the expression density. The relative coefficient was calculated with the formula below:
The Relative Coefficient=Cytokine expression density/β-actin expression density
Results:
I .The fluorescent quantitation RT-PCR detection demonstrated that five muscarinic receptor subtypes were all expressed to different extent at genetic level in normal human prostate.There was difference among these receptors' expression in different zones. It was mainly M_2, M_3, and M_1 receptors expressed.
In prefibromuscular zone, M_2 was obviously higher than other receptors(P<0.05),it is about three times of M_3's expression; The difference among M_3, M_1, M_5, M_4's expression was not statistically significant(P>0.05).
In transitional zone, M_2's expression was the highest (P<0.0001) in all the 5 receptors ,it is about 3 times of M_3's.M_3's was obviously higher than M4's(P<0.05).
In central zone, M_2's expression was the highest, but it was only statistically significant when it was compared with M4(P<0.05);the differences among M_2, M_1, M_3 and M_5 were not statistically significant(P>0.05).
In peripheral zone there was no significant difference among those receptor subtypes (P>0.1).
II .Western blot showed that there was protein expression difference among M_1, M2, M3 in normal, hyperplastic and cancerous prostatic tissue. In normal human prostate M_1 receptor expressed the highest(P<0.01). The differences of M3 receptor's expression in normal and pathological prostate tissue were statistically significance (P<0.05),while M_2 and M_1's wasn't(P>0.05).The highest expression receptor in cancerous prostate tissue was M3 receptor(P<0.01).
III. RT-PCR demonstrated that there was gene expression difference among M3 receptor, VEGF, endostatin in normal, hyperplastic and cancerous prostate tissue.M3 and VEGF's expression was higher in cancerous prostate than in normal prostate with significant difference (P<0.01).But endostatin's expression difference among the three different kinds of tissue wasn't statistically significant(P>0.05).It has positive correlation between M_3 and VEGF's expression (P<0.001) .
IV .The immunohistochemistry revealed that there were protein expression differences of VEGF and endostatin in normal, hyperplastic and cancerous prostatic tissue. VEGF and endostatin's expression were higher in prostate cancer tissue than in BPH tissue, while their expression were lowest in normal prostate tissue with the significant differences (P<0.01). In prostate cancer the more the Gleason score was,the more higher VEGF and endostatin were expressed (P<0.05) .Furthermore the expression in the late clinical stage wre higher than in the early stage (P<0.05) .
V .The Western blot indicated that VEGF and endostatin expressed highest in prostate cancer tissue ,moderate in BPH tissue, lowest in normal prostatic tissue with the significant differences (P<0.05 ) . It has positive correlation between VEGF and endostatin's expression (P<0.001) .
Conclusions:
I .There are genetic expressions of all the five muscarinic receptor subtypes in each zone of prostate, mainly are M_2, M_3, M_1.
II .In normal human prostate, the genetic expression of M_2, M_3, M_1 is similar to their protein expression,M3 receptor's protein expression is higher in cancerous than in normal prostatic tissue, which means it concerned with tumorigenesis of prostate .
III.M_3, VEGF and endostatin's expression in prostatic cancer is related with the tumor's clinical stage and pathological Gleason grading.
IV. It has positive correlation between M_3, VEGF and endostatin's expression in human prostate tissue.M_2, M_3, VEGF and endostatin's expression is high in prostatic cancer tissue.
V.M_3, VEGF, ES, and CD34 are related with genesis and development of prostatic tumor. They can be used as new targets of diagnosis and treatment for prostastic tumor.
前列腺是成年男性的重要器官，具有分泌前列腺液、协助射精、排尿等多种重要功能。前列腺生理功能的紊乱及病理变化可以导致许多疾病的发生，严重影响男性的身心健康。良性前列腺增生(BPH)是我国男性发病率最高的良性肿瘤，60岁以上老年人有50％发生良性前列腺增生症也是最常见的老年病致死原因，前列腺癌(PCa)一直是西方国家男性最常见的恶性肿瘤。在我国尽管前列腺癌的发病率相对较低，但近几年也有逐年上升的趋势。对前列腺癌进行早期发现和有效的治疗已使总体死亡率有了明显的降低。然而，晚期前列腺癌的治疗仍然是一个难题。慢性前列腺炎由于其不适症状重、治疗效果不满意，影响着患者的正常生活，因而也一直困扰着国内外泌尿外科医生。前列腺疾病的危害如此之大足以引起我们的重视，然而前列腺这些疾病的发病机理很多还不清楚，治疗效果还很不满意，还需要进一步深入研究。
前列腺的生长与功能受多种神经系统的调节，包括胆碱能神经与去甲肾上腺素能神经，近年来对前列腺的组织结构与功能的研究已经越来越深入和广泛，特别是对调节前列腺功能的植物神经系统的研究显示前列腺疾病发生、发展与神经系统的调节关系密切。在良性前列腺增生症的治疗上α受体阻滞剂已经取得了巨大的成功，雄激素阻断也已经成功地用于前列腺癌的内分泌治疗。然而对于胆碱能M受体在前列腺生理功能与疾病发生发展中的作用还不是很清楚，也还没有这方面的药物应用于前列腺疾病的治疗。
认识胆碱能M受体在前列腺中的作用，并把胆碱能药物用于前列腺疾病的治疗，首要的问题是弄清楚M受体在前列腺组织的分布特征，目前在良性前列腺增生组织及前列腺肿瘤细胞系已有这方面的研究，但是在正常前列腺各区带的分布规律目前还很少有研究报道，可查的资料也是本课题组2006年关于3种M受体亚型在正常前列腺表达规律的研究报道。
最近几年，关于前列腺癌发生和进展中发生的分子生物学改变方面的知识增加迅速，对肿瘤分子生物学更好的理解对于建立新的对策来延缓或抑制肿瘤的进展具有重要的意义。
研究表明在许多肿瘤细胞都有胆碱能受体表达，并且表达强度高于正常。对乳腺癌及其他肿瘤细胞系的研究发现，M_3受体与肿瘤的发生、分级密切相关，胆碱能受体的活动通过不同的信号途径可以刺激细胞的增殖。既往的研究发现M_1、M_2、M_3能刺激细胞的生长与分化，而M_4的这种功能则不明显。并且起调节作用的主要是M_3受体。这说明M_3受体表达的变化在肿瘤发生中具有重要的作用，了解M_3受体在正常前列腺、良性前列腺增生、前列腺癌组织中的表达变化规律对于探讨其在前列腺肿瘤发生、发展中的作用及其分子生物学机制具有重要的意义。
在肿瘤的发生、发展过程中，肿瘤细胞的增殖与新生血管的形成是重要的环节，在乳腺癌的LMM3细胞系的研究发现，其主要受体亚型为M_3，且表达量是LM3细胞的40倍。胆碱能受体激动剂CARB能够刺激LMM3细胞的生长与新生血管生成，选择性的M_3受体拮抗剂pf-HHSiD能抑制这种作用，并且试验表明CARB能刺激VEGF-A的表达，选择性的M_3受体拮抗剂pf-HHSiD也能抑制这种作用。这说明M_3受体在LMM3细胞的生长与血管生成中起着十分重要的作用。目前关于M_3受体与良性前列腺增生、前列腺癌发生发展的关系方面的研究还很少，并且已有的研究也是在没有严格意义上的正常对照的基础上完成的。
血管生成受到正负两方面因子的调控：血管生成刺激因子和血管生成抑制因子。血管内皮生长因子(vascular endothelial growth factor，VEGF)和内皮抑素(Endostatin ES)分别是目前已知的最强的血管生成促进因子和抑制因子，联合检测二者在肿瘤组织中的表达有助于更加深入地了解促血管生成因素和抑制血管生成因素在血管生成和肿瘤浸润转移中的作用。
血管内皮生长因子是肿瘤血管新生的重要作用因子。在绝大多数恶性肿瘤中均呈现VEGF及其受体的高表达，以旁／自分泌作用机制，促进内皮细胞分裂、增殖、诱导血管新生，并可直接作用于肿瘤细胞，刺激肿瘤细胞生长。在前列腺内VEGF可由正常导管内皮细胞、腺癌细胞和浸润的淋巴细胞合成。
VEGF的表达随着人类前列腺癌细胞的转移特性的增加而增加，而且恶性肿瘤细胞合成、分泌的VEGF刺激血管生成的效率更高。VEGF受体在前列腺肿瘤血管内皮细胞和肿瘤细胞上均有表达，这说明VEGF除了以旁分泌刺激血管内皮细胞增殖外，还能通过自分泌调节肿瘤细胞增殖。
Endostatin是从血管内皮瘤培养液中分离出的18型胶原蛋白C端氨基酸片段。它可特异性地抑制内皮细胞增殖、迁移和肿瘤血管生成，诱导细胞凋亡，使肿瘤原发灶及转移灶处于休眠状态，停止生长。
文献报道肿瘤内微血管密度(microvessel density MVD)反映了肿瘤间质血管生成的强度，随着MVD的增高，肿瘤的复发呈上升趋势。目前MVD已经成为评价肿瘤组织内新生血管形成程度的重要指标。
研究前列腺组织中VEGF与内皮抑素、M_3受体三者在前列腺正常组织与肿瘤组织中的表达关系对于研究M_3受体在前列腺肿瘤发生发展中的作用，对于深入研究前列腺肿瘤的发生与发展机制以及对于晚期肿瘤治疗靶点的选择均具有重要的意义，此研究在人体前列腺肿瘤还未查见相关报道。
材料与方法：
本研究分两部分进行，第一部分，首先应用实时荧光定量RT-PCR方法对M受体及其各亚型在正常前列腺各区带的mRNA表达进行研究，探讨各亚型在各区带的基因表达差异，然后进一步对在前列腺组织表达较高并与肿瘤发生密切相关的三种受体亚型(M_1、M_2、M_3)通过Western blot方法检测其在正常前列腺、良性前列腺增生、前列腺癌组织中的表达差异，获得各亚型在不同前列腺组织中的蛋白表达特征，分析不同亚型在前列腺正常功能发挥与疾病发生、发展中的作用。第二部分，先通过RT-PCR方法研究M_3、VEGF、ES在正常前列腺、良性前列腺增生、前列腺癌三种组织的基因表达，再应用免疫组化及Western blot两种方法并对比CD34的表达综合对比VEGF与内皮抑素在正常前列腺、良性前列腺增生、前列腺癌三种组织中的蛋白表达差异，分析这两种物质的表达在前列腺肿瘤发生、发展中的作用及与前列腺癌临床分期、gleason评分的关系：最后通过与M_3受体在正常前列腺与良性前列腺增生、前列腺癌中的表达进行对比研究，进一步探讨M_3受体在前列腺肿瘤发生、发展中的作用。这对于以后前列腺疾病治疗药物靶点的开发具有重要的意义。
取材：
RT-PCR及Western blot检测采用新鲜标本，其中正常前列腺组织采用健康成人新鲜尸体前列腺标本，良性前列腺增生、前列腺癌标本为采用前列腺切除手术或穿刺活检取得的标本(取自山东大学第二医院及渮泽市立医院2003-2006年的住院病人)，所有标本取出后均放于液氮中，转-80℃冰箱保存，实验前取出复温。检测M受体亚型在前列腺各区带的表达所用正常前列腺标本7例，检测M_3受体、VEGF、内皮抑素基因与蛋白表达所用标本分别为：正常前列腺36例、良性前列腺增生36例、前列腺癌8例。
免疫组化标本采用石蜡包埋标本，正常前列腺组织来自健康、新鲜尸体前列腺标本，良性前列腺增生、前列腺癌标本为采用前列腺切除手术或穿刺活检取得的标本(取自山东大学第二医院2002-2006年的住院病人)，标本经10％福尔马林浸泡，石腊包埋。其中正常前列腺36例，良性前列腺增生36例，前列腺癌36例。
操作在超净台进行，取新鲜标本时，每取一个部位均更换手术刀片，正常前列腺组织每个标本取相邻3块，分别做RT-PCR与Western blot及做石腊块用于免疫组化。
实验方法：
一、实时荧光定量RT-PCR方法检测正常成人前列腺各区带M受体各亚型(M_1～M_5)基因表达
实验所用物品的去RNase处理，将组织低温下研碎，组织提取总mRNA，合成cDNA，进行PCR扩增反应，荧光定量RT-PCR反应条件：95℃10s，95℃5s，60℃45s，40个循环；得到每个部位、每个受体的Ct值，用△Ct法转换为其初始相对浓度。其换算公式为：△Ct=Ct_(样本)-Ct_(β-actin)，平均相对含量=2~(-△Ct)。
统计学处理
应用SAS9.0软件进行统计学处理，统计方法为配伍组设计资料的方差分析。并应用q检验进行组间两两比较，P＜0.05认为差异有统计学意义。
二、RT-PCR方法检测M_3受体、VEGF、内皮抑素在正常前列腺、良性前列腺增生、前列腺癌组织中的基因表达
实验所用物品的去RNase处理，组织低温下研碎，提取总RNA，进行逆转录(RT)反应合成cDNA。聚合酶链反应(PCR)。定量分析：将凝胶电泳图像输入美国Kodak凝胶分析系统，应用1 D Image Analysis Software进行表达强度分析，按下公式计算相对系数。
相对系数=细胞因子表达强度／β-actin表达强度
统计学处理
应用SAS9.0软件进行统计学处理，统计方法为随机设计资料的方差分析。应用q检验进行组间两两比较，P＜0.05认为差异有统计学意义。M_3与VEGF采用pearson相关性分析。
三、免疫组化法检测VEGF、内皮抑素、CD34在正常前列腺、良性前列腺增生、前列腺癌组织中的蛋白表达
首先将蜡块连续切片4片，片厚4μm。取胎盘组织以PBS代替一抗做阴性对照，把每组标本的3片分别做VEGF、内皮抑素及CD34免疫组化，另一切片做HE染色。根据HE染色，对每个样本进行病理学审查(由病理科医生协助完成)，排除病变样本。结果判定以染色阳性物质呈棕黄色。M_3、VEGF、内皮抑素染色结果判定：根据阳性细胞显色深浅及阳性细胞百分率分级：①显色深浅：不显色0分；浅黄色1分；棕黄色2分；深褐色3分。②阳性细胞百分率：无阳性细胞显色0分；阳性细胞显色＜25％为1分；25％～50％为2分；阳性细胞显色＞50％为3分。二者得分相加：0分—阴性(-)；1～2分—弱阳性(+)；3～4分—阳性(++)；5～6分—强阳性(+++)。结果判断在双盲下进行，每张切片由两位病理医师分别阅片记数。CD34染色阳性的单个内皮细胞或一簇内皮细胞作为一个微血管，在200×视野下计数3个血管密度最高区域(热点)的微血管数目，其平均值即为肿瘤的微血管密度(MVD)。
统计学处理
应用SAS9.0软件进行统计学处理，统计方法为随机设计资料的方差分析与x~2检验，P＜0.05认为差异有统计学意义。M_3与VEGF采用pearson相关性分析。
四、Western blot方法检测三种M受体亚型(M_1、M_2、M_3)、VEGF、内皮抑素的蛋白表达
取新鲜组织块，称量后提取蛋白(M受体提取膜蛋白、VEGF、内皮抑素提取胞浆蛋白)、加上养液、煮沸、在Bio-Rad垂直电泳仪中灌注质量分数10％分离胶和质量分数7.5％浓缩胶，加入不同组样本，稳压120V电泳90min，然后在转膜仪上将凝胶上的蛋白转移到PVDF膜上，稳压100V转移60min，取出膜后用7％脱脂牛奶封闭60min，加入一抗，4℃过夜，次日加入抗兔二抗孵育2h，DAB显色，扫描图像，输入美国Kodak图像分析系统，应用1 D Image Analysis Software进行表达强度分析，按下公式计算相对系数。
相对系数=细胞因子表达强度／β-actin表达强度
统计学处理
应用SAS9.0软件进行统计学处理，统计方法为随机设计资料的方差分析。应用q检验进行组间两两比较，P＜0.05认为差异有统计学意义。VEGF与ES采用pearson相关性分析。
结果：
第一部分
1、荧光定量RT-PCR方法检测结果显示五种M受体亚型在正常成人前列腺均有不同程度的基因表达，各个区带各受体的表达存在差异。以M_2、M_3、M_1受体的表达为主。
在前纤维肌区M_2的表达明显高于其他受体(P＜0.05)，约是M_3表达量的3倍；M_3、M_1、M_5、M_4的表达差异无统计学意义(P＞0.05)。
在移行带M_2的表达明显高于其他受体(P＜0.0001)，约是M_3表达量的三倍。M_3的表达明显高于M_4(P＜0.05)。
在中央带M_2的表达最高，但只与M_4之间比较差异有统计学意义(P＜0.05)；M_2、M_1、M_3、M_5四者间差异无统计学意义(P＞0.05)。在外周带各受体亚型的表达差异均无统计学意义(P＞0.1)。
2、Western blot检测显示M_1、M_2、M_3在正常前列腺、良性前列腺增生、前列腺癌组织中的蛋白表达存在差异。
在正常前列腺M_2的表达高于M_3，更高于M_1，三者比较差异均具有统计学意义(P＜0.01)。
在良性前列腺增生与前列腺癌组织M_2、M_3受体的表达高于M_1，差异具有统计学意义(P＜0.01)。M_2、M_3受体之间的表达差异无统计学意义(P＞0.05)。在正常前列腺与病变前列腺组织中表达差异具有统计学意义。
M_1受体在正常前列腺、良性前列腺增生、前列腺癌三种组织的表达差异无统计学意义(P＞0.05)。
M_2受体在正常前列腺、良性前列腺增生、前列腺癌三种组织的表达差异无统计学意义(P＞0.05)。
M_3受体在前列腺癌组织的表达高于在良性前列腺增生组织的表达，正常前列腺组织的表达最低，差异均具有统计学意义(P＜0.01)。
第二部分
1、RT-PCR结果显示M_3受体、VEGF、内皮抑素在正常前列腺、良性前列腺增生、前列腺癌组织的基因表达存在差异
M_3受体在不同前列腺组织的基因表达存在差异，表达顺序为：前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义(P＜0.01)。
VEGF在不同前列腺组织的表达不同，表达顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义(P＜0.01)。
内皮抑素在不同前列腺组织的表达不同，其均值顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均不具有统计学意义(P＞0.05)。
M_3受体的表达与VEGF的表达具有正相关关系(r=0.4999 P＜0.001)。
2、免疫组化结果显示在正常前列腺、良性前列腺增生、前列腺癌组织VEGF、内皮抑素的蛋白表达存在差异
CD34在不同组织的表达强度顺序为：前列腺癌＞良性前列腺增生＞正常前列腺(P＜0.05)。
MVD与前列腺癌临床分期的关系比较，因为样本含量少，合并临床Ⅰ、Ⅱ期与临床Ⅲ、Ⅳ期相比，P＞0.05，差异无统计学意义。
MVD与前列腺癌Gleason评分的关系比较，评分≥8分的与评分＜8份的比较，P＞0.05，差异无统计学意义。
VEGF在不同组织的表达强度顺序为：前列腺癌＞良性前列腺增生＞正常前列腺，差异均具有统计学意义(P＜0.01)。
ES在不同组织的表达强度顺序为：前列腺癌＞良性前列腺增生＞正常前列腺，差异均具有统计学意义(P＜0.01)。
VEGF与前列腺癌临床分期的关系比较，因为样本例数含量少，合并临床Ⅰ、Ⅱ期与临床Ⅲ、Ⅳ期相比，差异有统计学意义(P＜0.01)。
VEGF与前列腺癌Gleason评分的关系比较，评分≥8分的与评分＜8分的比较，差异有统计学意义(P＜0.05)。
ES与前列腺癌临床分期的关系比较，因为样本含量少，合并临床Ⅰ、Ⅱ期与临床Ⅲ、Ⅳ期相比，差异具有统计学意义(P＜0.01)。
ES与前列腺癌Gleason评分的关系比较，评分≥8分的与评分＜8分的比较，差异具有统计学意义。(P＜0.05)
3、Western blot检测结果显示VEGF、内皮抑素的蛋白表达存在差异
VEGF在不同前列腺组织的表达不同，表达顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义。(P＜0.05)
内皮抑素在不同前列腺组织的表达不同，其均值顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义(P＜0.05)。
VEGF的表达与内皮抑素的表达具有正相相关(r=0.5582 P＜0.001)。
结论：
1、前列腺各个区带均有不同程度的5种M受体亚型的基因表达，主要以M_2、M_3、M_1为主。
2、M_2、M_3、M_1受体在正常前列腺的蛋白表达与基因表达具有相同的趋势，M_3受体在前列腺肿瘤组织的蛋白表达高于在正常前列腺的表达，与前列腺肿瘤的发生、发展关系密切。
3、VEGF、ES与前列腺肿瘤的发生及前列腺肿瘤的临床分期、gleason评分有密切关系。
4、M_3、VEGF、ES在前列腺组织的表达具有正相直线相关关系。
5、M_3、VEGF、ES、CD34可进一步研究作为前列腺肿瘤诊断指标及治疗新靶点的选择。
Research background and objects:
Prostate which is an important organ of male adult has many important functions, such as prostatic fluid secretion, ejaculation assisting, urination and so on. The physiological and functional disorder of the prostate and its pathological changes can cause many diseases, even affect the physical and mental health of men. Benign prostatic hyperplasia is,of the highest incidence disease among tumors of men in our country. Prostate cancer is one of the most common malignant tumors for men in western country. Although the incidence of prostate cancer is low in our country, it has the tendency to increase in recent years. Chronic prostatitis affects patients' life because of severe symptoms and unsatisfied treatment, so that it continuously harasses urologists all over the world. Prostatic diseases' disadvantages are serious enough to call our attention, but the mechanism of these diseases is still unknown and the treatment is not so satisfied.
Detective and aggressive treatment of early prostate cancer resulted in a significant decline of overall mortality rates. However, the treatment of advanced tumor stages is still a crucial problem. Knowledge on molecular alterations taking place in the development and progression of prostate cancer increased rapidly in the past few years, thanks also to the use of novel techniques that enabled to perform broad gene expression profiling of tumors. This better understanding of the molecular biology of prostate cancer led to the establishment of novel strategies to delay/inhibit tumor progression.
The growth and function of prostate is regulated by many nervous systems, including cholinergic and epinephrine nerves systems. In the past few years the construction and function of prostate have been deeply researched. The researches of autonomic nerve system's regulation for function of the prostate demonstrated that the regulation of nerve system was intimately related to the genesis and development of prostatic diseases. Great success has been obtained in the research of alpha receptor inhibitor used in the treatment of benign prostatic hyperplasia, and androgen-blocking has been successfully used in the endocrine treatment of prostate cancer. But cholinergic muscarinic receptor's functions in regulating the prostatic physiologic function, disease genesis and development is still unclear. And also there has been no such kind of drugs used in the treatment of prostatic diseases.
In order to better understand the roles of cholinergic muscarinic receptors in prostate and to apply cholinergic drugs in the treatment of prostatic diseases, we should make out the distribution characteristics of muscarinic receptor in normal human prostatic tissue. Up to now there are only researches about muscarinic receptor on benign hyperplastic prostate tissue and prostatic tumor cell lines, but the report of distribution regularity in different zones of normal human prostate is only fewer. The only report is done by our research team about the distribution regularity of the three kinds of muscarinic receptor subtypes in normal human prostate in 2006
The cholinergic receptor expression was prevalent in many tumors, in which mostly higher than in the normal tissue. The research about breast carcinoma and other tumors revealed that M_3 receptor was intimately related with the carcinogenesis and classification of tumor. The activity of cholinergic receptor could provoke the cell proliferate through different signal pathway. The previous research found out that M_1, M_2. M_3 receptors could stimulate cell growth and differentiation, but M_4 was not found having such funetions. Furthermore M_3 receptor is the main subtype to play the role of regulator, and be changed much in tumor generation. To understand the expression rule of M_3 receptor in healthy, hyperplastic, cancerous prostate tissue is very important for making clear the role of M_3 receptor and its molecular biologic mechanism in genesis and development of prostatic tumor.
The proliferation of tumor cells and the formation of new blood vessels were important steps in the process of the genesis and development of tumor. In the research of breast cancer LMM3 cell line, it was found that the main subtype was M3 and its expression quantity was 40 folds of LM3 cell. The cholinergic receptor agonist CARB could provoke the growth and neovascular genesis of LMM3 cell, but this function could be suppressed by the selective M3 receptor antagonist. It was also demonstrated that CARB could promote the expression of VEGF-A, which could be inhibited by selective M3 receptor antagonist pf-HHSiD. All these findings told us that M3 receptor played an important role in LMM3 cell growth and neovascular genesis. But the researches about M 3 receptor in prostatic hyperplasia and cancer was much fewer, and the fewer finished study was done based on not strict normal control.
Vascular endothelial growth factor(VEGF) is an important factor in neogenesis of tumor vessels. VEGF and its receptor are highly expressed in most malignant tumors, through para/autoendocrine reaction, promoted endothelial cell division, proliferation and induced vascular neogenesis, directly affecting tumor cell and provoking it to grow. In prostate, VEGF could be synthesized by normal duct endothelial cell, adenoma cell and infiltrative lymphocyte.
The stronger the metastatic characteristic of human prostatic cancer cell was, the higher VEGF expression would be, and neovascular genesis would be more effectively promoted by VEGF which was synthesized and secreted by malignant cell. The VEGF receptor expressed on both the vessel endothelium of prostatic tumor and the tumor cell showed that VEGF not only stimulated vascular endothelial proliferation by paraendocrine way but also regulated tumor cell proliferation by autoendocrine way.
Endostatin was XVIII collagen C-terminal amino segment which was isolated from culture solution of vascular endothelioma. It could specifically inhibit endothelial cell proliferation, metastasis and tumorous angiogenesis, and could induce cell apoptosis, so that it made the primary and metastatic site of tumor stay in dormant state and stop growing.
To study VEGF, endostatin and M_3 receptor's relation in normal and tumor prostate is very important for further understanding the mechanism of tumorigenesis,development, and selecting the target of treatment in late stage tumor. At present the research on this aspect about human prostatic tumor has not been reported.
For the above reason my research firstly studied the mRNA expression of muscarinic receptor subtypes in each zone of normal prostate and discussed the expression difference of each subtype among different zones. Then we detected the expression of those three kinds of receptors, among the normal, hyperplastic and cancerous prostate, using Western blot, which were highly expressed in the prostatic tissue and had intimate relationship with the tumor genesis. We gained the protein expression difference of each subtype in different zones of prostate tissue and analyzed the role of different subtype in normal human prostate,and in the genesis.developmeiit of diseases. At last using RT-PCR, immunohistochemistry, Westsern blot, we comparatively studied the expression of VEGF and endostatin in prostatic tissue at genetic and protein levels, together with the expression of M_3 receptor in normal, hyperplastic and cancerous prostate, and discussed their roles in genesis and development of prostatic tumor.
These researches were important for understanding M receptor, VEGF and endostatin's roles in prostate's function educing and disease generation, also it is more important for developing new drugs for prostatic diseases .
Material and method:
Materials:
The fresh normal prostates were obtained from healthy adult donators who were died of accident. The samples of hyperplastic, cancerous prostates were taken from the hospitalized patients in The Second Hospital of SDU from 2000 to 2006, and from prostatectomy or core biopsy. All the tissues were put in nitrogen liquid and kept in -80℃refrigerator for further using. Seven samples of normal prostate were used in detecting M receptor subtype in each zone of prostate. The specimens used in detecting M_3 receptor, VEGF, endostatin at genetic and protein levels included 36 normal prostates, 36 hyperplastic ones, and 8 cancerous ones.
The samples detected with immunohistochemistry were paraffin-embedded ones. The normal prostatic tissue was from fresh corpses of healthy adults. The hyperplastic, cancerous samples were from hospitalized patients of The Second Hospital of SDU from 2000 to 2006, taken with prostatectomy or care biopsy. The samples were soaked in 10% formalin and paraffin-embedded. There were 36 normal prostate, 36 hyperplastic ones and 36 cancerous ones. All the operations were taken on super clean bench. During taking fresh samples, the operating knife blade should be changed for different site. Every sample of normal prostate was taken out 3 pieces, respectively used in RT-PCR,western blot and made into paraffin-embedded block which would be used in immunohistochemistry.
Methods:
I .Detect muscarinic receptor subtypes'genetic expression in each zone of normal human prostate with real-time fluorescent quantitative RT-PCR
Process the experiment material with RNase. Levigate the tissue at low temperature. Extract the total mRNA. Synthesize cDNA. Carry out PCR amplification reaction. Real Time PCR reaction condition: 95℃10s, 95℃5s, 60℃45s, 40 cycles; Every receptor's threshold cycle(Ct) value of each site which we got was transformed into its initial relative concentration with△Ct way. The transformation formula:△Ct=Ct_(sample)-Ct_(β-actin). The mean relative quantity = 2~(-△Ct) . Apply SAS9.0 software in statistical treatment. The statistical method was variance analysis to pair-matched data. And q test was used to compare every two groups. The difference was considered as statistically significant when P<0.05.
II .Using RT-PCR detect M_3 receptor, VEGF, endostatin's expression in normal,hyperplastic, cancerous prostate at genetic level:
The experiment material was processed with RNase. Levigate the tissue at low tempreture. Extract the total RNA.The RNA was then reverse transcribed into cDNA. Input gel electrophoresis picture to the American Kodak gel analystic system.Use 1 D Image Analysis Software to analyse the expression intensity.Calculate the relative coefficient with the formula below.
The relative coefficient=cytokine expression intensity/β-actin expression intensity
III. Detect VEGF, endostatin, CD34's expression in normal, hyperplastic, and cancerous prostate with immunohistochemical method
Firstly we cut 4 serial sections with thickness 4 micrometres from the paraffin block. Fetal tissue with PBS instead of first Ab serves as negative control. The 3 slices of every group were respectively used in VEGF , endostatin and CD34's immunohistochemical detection. The other one was HE stained. Every sample was inspected by the pathologist. In the result evaluation the positive stained should be brown. M_3, VEGF, endostatin's staining result assessment: to classify them according to the extent of positive cell coloration and positive cell percentage: i according to the coloration:no color score 0;buff 1;buffy 2;sable 3. ii according to the positive cell percentage: no positive cell score 0; positive cell<25% 1; positive cell 25%-50% 2; positive cell>50% 3;Then add the two score : 0-negative(-);1-2-weakly positive(+); 3-4-positive(++);5 -6-strongly positive(+++).The result assessment was carried out with double blind method. Every slice was inspected and counted by two pathologists respectively. We considered one single endothelium or one cluster of endothelium, which was CD34 positive stained as one microvessel. We counted the microvessel number of 3 different visual field where the vessel density was the highest in 200X view field. The mean value was treated as tumor's microvessel density.
iv. Detect M_1, M_2, M_3, VEGF and endostatin's protein expression in normal, hyperplastic, cancerous prostate with western-blot method
Fresh tissue were taken and weighted.Then the protein(we extracted membrane protein for M receptor, extracted the cytoplasmic protein for VEGF and endostatin) was extracted and denatured.Different samples were added in Bio-Rad vertical electrophoresis apparatus which had been perfused with weight percentage 10% separating gel and weight percentage 7.5% stacking gel, and ran for 90 min at stable voltage 120 volt. Then buffer transferred the protein in the gel onto PVDF membrane with transferring membrane, at stable voltage 100 volt for 60 min. The membrane was taken out and blocked by 7% skimmed milk for 60 min. Then it was incubated with first Ab overnight at 4℃, washed thoroughly and with the second Ab for 2 hours. Stained the membrane with DAB and scanned the picture. The picture was inputted into the American Kodak Image Analystc System. Using 1 D Image Analysis Software analyzed the expression density. The relative coefficient was calculated with the formula below:
The Relative Coefficient=Cytokine expression density/β-actin expression density
Results:
I .The fluorescent quantitation RT-PCR detection demonstrated that five muscarinic receptor subtypes were all expressed to different extent at genetic level in normal human prostate.There was difference among these receptors' expression in different zones. It was mainly M_2, M_3, and M_1 receptors expressed.
In prefibromuscular zone, M_2 was obviously higher than other receptors(P<0.05),it is about three times of M_3's expression; The difference among M_3, M_1, M_5, M_4's expression was not statistically significant(P>0.05).
In transitional zone, M_2's expression was the highest (P<0.0001) in all the 5 receptors ,it is about 3 times of M_3's.M_3's was obviously higher than M4's(P<0.05).
In central zone, M_2's expression was the highest, but it was only statistically significant when it was compared with M4(P<0.05);the differences among M_2, M_1, M_3 and M_5 were not statistically significant(P>0.05).
In peripheral zone there was no significant difference among those receptor subtypes (P>0.1).
II .Western blot showed that there was protein expression difference among M_1, M2, M3 in normal, hyperplastic and cancerous prostatic tissue. In normal human prostate M_1 receptor expressed the highest(P<0.01). The differences of M3 receptor's expression in normal and pathological prostate tissue were statistically significance (P<0.05),while M_2 and M_1's wasn't(P>0.05).The highest expression receptor in cancerous prostate tissue was M3 receptor(P<0.01).
III. RT-PCR demonstrated that there was gene expression difference among M3 receptor, VEGF, endostatin in normal, hyperplastic and cancerous prostate tissue.M3 and VEGF's expression was higher in cancerous prostate than in normal prostate with significant difference (P<0.01).But endostatin's expression difference among the three different kinds of tissue wasn't statistically significant(P>0.05).It has positive correlation between M_3 and VEGF's expression (P<0.001) .
IV .The immunohistochemistry revealed that there were protein expression differences of VEGF and endostatin in normal, hyperplastic and cancerous prostatic tissue. VEGF and endostatin's expression were higher in prostate cancer tissue than in BPH tissue, while their expression were lowest in normal prostate tissue with the significant differences (P<0.01). In prostate cancer the more the Gleason score was,the more higher VEGF and endostatin were expressed (P<0.05) .Furthermore the expression in the late clinical stage wre higher than in the early stage (P<0.05) .
V .The Western blot indicated that VEGF and endostatin expressed highest in prostate cancer tissue ,moderate in BPH tissue, lowest in normal prostatic tissue with the significant differences (P<0.05 ) . It has positive correlation between VEGF and endostatin's expression (P<0.001) .
Conclusions:
I .There are genetic expressions of all the five muscarinic receptor subtypes in each zone of prostate, mainly are M_2, M_3, M_1.
II .In normal human prostate, the genetic expression of M_2, M_3, M_1 is similar to their protein expression,M3 receptor's protein expression is higher in cancerous than in normal prostatic tissue, which means it concerned with tumorigenesis of prostate .
III.M_3, VEGF and endostatin's expression in prostatic cancer is related with the tumor's clinical stage and pathological Gleason grading.
IV. It has positive correlation between M_3, VEGF and endostatin's expression in human prostate tissue.M_2, M_3, VEGF and endostatin's expression is high in prostatic cancer tissue.
V.M_3, VEGF, ES, and CD34 are related with genesis and development of prostatic tumor. They can be used as new targets of diagnosis and treatment for prostastic tumor.
引文
1. Kevin T. Mc Vary. Prostate innervation. Prostate 1998;Suppl 8:2-13
2. Dail WG: Autonomic innervation of male reproductive genitalia.In Maggi CA (ed): "Nervous Control of the Urogenital System," Switzerland: Harwood Academic Publishers, 1993:69-101.
3. J .S. Dixon, P.Y.P. Jen, J.A. Gosling, The distribution of vesicular acetylcholine transporter in human male genitourinary organs and its co-localization with neuropeptide Y and nitric oxide synthase, established. Neurourol. Urodyn. 2000:19 185-194.
4. Irving Nadelhaft C holinergic axons in the rat prostate and neurons in the pelvic ganglion Brain Res 2003: 989 52-57
5. Chappie, C. R., Crowe, R., Gilpin, S. A., Gosling, J., & Burnstock, G. The innervation of the human prostate gland—the changes associated with benign enlargement. J Urol 1991:146, 1637- 1644.
6. Gosling JA: Autonomic innervation of the prostate. In Hinman F Jr (ed): "Benign Prostatic Hypertrophy," New York: Springer- Verlag, 1983:349-360.
7. Wang J-M, McKenna KE, Lee C: Determination of prostatic secretion in rats: Effect of neurotransmitters and testosterone. Prostate 1991;18:289-301.
8. Rimmaudo LE,de la Torre E,Sacerdote de Lustiq E,et ai.Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis. Biochem Biophys Res Commun,2005,334(4): 1359-64.
9. Cheng K, Chen Y , Zimniak P,et al. Functional interaction of lithocholic acid conjugates with M3 muscarinic receptors on a human colon cancer cell line. Biochimica et Biophysica Acta, 2002,1588 (1) : 48- 55.)
10. Rimmaudo L E,de la Torre E,Sacerdote de Lustig E,et al. Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis[J].Biochem Biophys Res Commun,2005,334(4): 1359-64.
11. Folkman J . What is the evidence that tumors are angiogenesis dependent. J Natl Cancer Inst, 1990 ;82 (1) :4-6
12. 赵利枝,杨日芳,汪海. 内皮细胞与血管形成. 中国药理学通报,2002;18(4):361-4
13. Ferrer FA ,Miller LJ ,Andrawis RI ,et al. J Urol ,1997 ; 157 :2329-2333
14. Freeman MR ,Schneck FX,Gagnon ML ,et al . Cancer Res ,1995 ;55 :4140-4145
15. Chen HJ, Treweeke AT ,YQ Ke ,et al .Br J Cancer ,2000 ;82 :1694-1701
16. Schuch G, Kisker O, Atala A, et al. Pancreatic tumor growth is regulated by the balance between positive and negative modulators of angiogenesis [J]. Angiogenesis, 2002, 5(3): 181-190.
17. Robak E, Wozniacka A, Sysa-Jedrzejowska A, et al. Circulating angiogenesis inhibitor enodostatin and positive endothelial growth regulators in patients with systemic lupus erythematosus [J]. Lupus, 2002, 11(6):348-355.
18. Chappie C R, Crowe R, Gilpin S A, et al. The innervation of the human prostate gland—the changesassociated with benign enlargement[J]. J Urol, 1991,146(6): 1637-1644.
19. Hedlund P, Ekstrom P, Larsson B, et al. Heme oxygenase and NO-synthase in the human prostate—relation to adrenergic, cholinergic and peptide-containing nerves[J]. J Auton Nerv Syst, 1997,63(3): 115-126.
20. Dail W G. Autonomic innervation of male reproductive genitalia.In C. A. Maggi (Ed.), Nervous Control of the Urogenital System[M]. Switzerland: Harwood Academic Publishers, 1993,69-101
21. Sjostrand N O, Ehren I, Eldh J,et al. NADPH diaphorase in glandular cells and nerves and its relation to acetylcholinesterase- positive nerves in the male reproductive tract of man and guinea-pig[J]. Urol Res,1998,26(3):181-188.
22. Dixon J S, Jen P Y,Gosling J A. The distribution of vesicularacetylcholine transporter in the human malegenitourinary organsand its co-localization with neuropeptide Y and nitric oxide synthase[J].Neurourol Urodyn,2000,19(2): 185-194.
23. Pontari M A, Luthin G R, Braverman A S, et al. Characterization of muscarinic cholinergic receptor subtypes in rat prostate[J]. J Recept Signal Transduct Res,1998,18(2-3):151-166.
24. Hedlund H, Andersson K E, Larsson B. Alpha-adrenoceptors and muscarinic receptors in the isolated human prostate[J]. J Urol,1985,134(6):1291-1298.
25. Caulfield M P. Muscarinic receptors-characterization, couplingand function[J]. Pharmacol Ther,1993,58(3):319-379.
26. Luthin G R, Wang P, Zhou H, et al. Role of ml receptor-G protein coupling in cell proliferation in the prostate[J]. Life Sci ,1997,60(13-14):963-968.
27. Braverman A S ,Tallarida RJ,Ruggieri M R. Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction [J]. Am J Physiol Regul Integr Comp Physiol. 2002 ,283(3):R663-668.
28. Tayebati S K, El-Assouad D, Ricci A,et al. Immunochemical and immunocytochemical characterization of cholinergic markers in human peripheral blood lymphocytes[J]. J Neuroimmunol. 2002,132(l-2):147-155.
29. Ruggieri M R, Colton M D, Wang P, et al. Human prostate muscarinic receptor subtypes[J]. J Pharmacol Exp Ther,1995,274(2):976-982.
30. Fernandez J L, Rivera L, Lopez P G, et al. Characterization of the muscarinic receptor mediating contraction of the dog prostate[J]. J Auton Pharmacol, 1998, 18(4):205-211.
31.Eglen R M, Hegde S S, Watson N. Muscarinic receptorsubtypes and smooth muscle function[J]. Pharmacol Rev,1996,48(4):531-565.
32. Ehlert F J, Ostrom R S, Sawyer G W. Subtypes of muscarinicreceptor in smooth muscle[J]. Life Sci,1997,61(18): 1729-1740.
33. Ehlert F J, Sawyer G W, Esqueda E E. Contractile role ofM2 and M3 muscarinic receptors in gastrointestinal smooth muscle[J]. LifeSci,1999,64(6-7):387-394.
34. Lau W A, Pennefather J N. Muscarinic receptor subtypes in the rat prostate gland[J]. Eur J Pharmacol.1998,343(2-3): 151-156.
35. Obara K, Arai K, Miyajima N, et al. Expression of m2 muscarinic acetylcholine receptor mRNA in primary culture of human prostate stromal cells[J]. Urol Res,2000,28(3): 196-200.
36. Mottet N, Costa P, Bali JP. Autonomic nervous system and prostatic physiology. Specific features of the alpha-adrenergic system[J]. Prog Urol, 1999,9(1):26～36.
37．赵升田 袁明振 王洪伟等．M受体亚型在正常成人前列腺组织中的分布．山东大学学报，2006，44(3)：287-290
38. Rayford W, Noble M J, Austenfeld M A, et al. Muscarinic cholinergic receptors promote growth of human prostate cancer cells[J]. Prostate, 1997,30(3): 160～166.
39. Wessler I, Kirkpatrick C J, Racke K. The cholinergic 'pitfall': acetylcholine, a universal cell molecule in biological systems, including humans[J]. Clin Exp Pharmacol Physiol, 1999,26(3): 198～205.
40. Rayford W, Noble M J, Austenfeld M A, et al. Muscarinic cholinergic receptors promote growth of human prostate cancer cells. Prostate[J]. 1997,30(3): 160～166
41. Sydorenko V, Shuba Y, Thebault S. Receptor-coupled, DAG-gated Ca2+-permeable cationic channels in LNCaP human prostate cancer epithelial cells[J]. J Physiol,2003,548(Pt 3):823～836.
42. Blanco M, Robinson M J. Muscarinic M3 acetytcholine receptor immunostaining in paraffin-embedded normal and neoplastic prostatic gland tissue[J]. Ann Diagn Pathol,2004,8(6):333～336.
43. Borovikova L V, Ivanova S, Zhang M, et al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin [J]. Nature, 2000,405 (6785): 458-462.
44. Kendall M D, al-Shawaf A A. Innervation of rat thymus gland [J]. Brain Behav Immun, 1991, 5(1): 9-28.
45. Gushchin G V, Jakovleva E E, Kataeva G V. Muscarinic cholinergic receptors of rat lymphocytes: effect of antigen stimulation and local brain lesion [J] Neuroimmuno2 modulation, 1994,1 (4): 2592264.
46. Kuo Y, Lucero L, Michaels J, et al. Differential expression of nicotinic acetylcholine receptor subunits in fetal and neonatal mouse thymus [J]. J Neuroimmunol,2002,130(1-2): 140-154.
47. Fujino H, Uehara T, Murayama T, et al. Extracellular signal regulated protein kinase and c2jun N2terminal kinase are involved in ml musearinic receptor2enhanced interleukin22 production pathway in J urkat cells[J]. Biol Pharm B ull, 2000,23(10): 1198-1205.
48．姜建兰，邱一华，彭聿平．乙酰胆碱对自然杀伤细胞活性的影响．中国应用生理学杂志，2005，21(3)，330-332
49. Ferrara N,Houck K,Jakeman L,et al. The vascular endothelial growth factor family of polypeptides [J]. J Cell Biochem, 1991,47(3):211-218
50. Waltenberger J,Welsh LC,Claesson-Siegbahn A,et al. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor[J]. The J Biol Chem,1994,269(43):26988～26995
51. Ferrer FA,Miller LJ, Lindoquist R,et al. Expression of vascular endothelial growth factor receptors in human prostate cancer[J]. Urology,1999,54 (3):567～572
52. Ergun S,Kilic N,Fiedler W,et al. Vascular endothelial growth factor and its receptors in normal human testicular tissue[J]. Mol Cell Endocrinol,1997,131(1):9～20
53. Walther MM,Figg WD,Lineham WM. Intravesical suramin: a novel agent for the treatment of superficial transitional-cell carcinoma of the bladder[J]. World J Urol,1996,14 (suppl1):S8-11
54. Melnyk O,Zimmerman M,Kim KJ,et al. Neutralizing anti-vascular endothelial growth factor antibody inhibits further growth of established prostate cancer and metastases in a pre-clinical model[J]. J Urol, 1999,161 (3):960-963
55. Connolly JM,Rose DP. Angiogenesis in two human prostate cancer cell lines with differing metastatic potential when growing as solid tumors in nude mice[J]. J Urol, 1998,160(3pt1):932～936
56. Hahn D,Simak R,Steiner GE,et al. Expression of the VEGF-receptor Flt-1 in benign, premalignant and malignant prostate tissues[J]. J Urol, 2000,164(2):506～510
57. Latil A,Bieche I,Pesche S,et al. VEGF overexpression in clinically localized prostate tumors and neuropilin-1 overexpression in metastatic forms[J]. Int J Cancer,2000,89(2): 167～171
58. Gaqnon ML,Bielenberg DR, Gechtman Z, et al. Identification of a natural soluble neuropilin-1 that binds vascular endothelial growth factor: In vivo expression and antitumor activity[J]. Proc Natl Acad Sci USA , 2000 , 97(6) :2573-2578
59. Duque JLF ,Loughlin KR ,Adam RM,et al . Plasma levels of vascular endothelial growth factor are increased in patients with metastatic prostate cancer[J]. Urol ,1999 ,54(3) :523- 527
60. Mazzucchelli R ,Montironi R ,Santinelli A ,et al . Vascular endothelial growth factor expression and capillary architecture in high-grade PIN and prostate cancer in untreated and androgen-ablated patients[J]. Prostate ,2000,45(1) :72—79
61. Haggstrom S ,Bergh A ,Damber JE ,et al . Vascular endothelial growth factor content in metastasizing and nonmetastasizing Dunning prostatic adenocarcinoma[J]. Prostate ,2000,45(1) :42-50
62. Liu XH ,Kirschenbaum A ,Yao S ,et al . Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo[J]. J Urol ,2000 ,164(3 Pt l):3820-3825
63. Gaddipati JP ,Mani H ,Shefali ,et al . Inhibition of growth and regulation of IGFs and VEGF in human prostate cancer cell lines by shikonin analogue 93/637 (SA) [J]. Anticancer Res ,2000 ,20(4) :2547-2552
64. Viglietto G,Romano A ,Maglione D ,et al. Neovascularization in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not the placenta-derived growth factor. Oncogene[J] ,1996 ,13(3) :577-587
65. O'Reilly MS ,Boehm T ,Shing Y,et al . Endostantin :an endonous inhibitor of angiogenesis and tumor growt h [ J ]. Cell, 1997 , 88(2) :227 - 285
66. Miosge N , Sasaki T , Timpl R , et al . Angiogenesis inhabitorendostantin is a distinct component of elastic fibers in vessel walls[J ] . FASEB J ,1999 ,13 (13):1743- 1750
67. Sasaki T ,Fukai N ,Mann K,et al. St ruct ure ,function and tissue forms of t he C -terminal globular domain of collegen X VIII containning the angiogenesis inhabitor endostantin [ J ]. EMBO J , 1998 ,17 (15) :4249 - 4256
68. Chen W, Fu J, Liu Q, Ruan C, Xiao S. Retroviral endostatin gene transfer inhibits human colon cancer cell growth in vivo[J]. Chin Med J (Eng1), 2003, 116(10): 1582～1584.
69. Mi J, Sarraf-Yazdi S, Zhang X, Cao Y, Dewhirst MW,Kontos CD, Li CY, Clary BM. A comparison of antiangiogenic therapies for the prevention of liver metastases[J]. J Surg Res, 2006, 131 (1): 97～104.
70．骆成玉，赵丹宁，李世拥，杨晓明，梁龙，祝学光．内皮抑素对结肠癌肝转移的干预作用[J]．中华外科杂志，2001，39(3)：335～337．
71. Folkman J. Antiangiogenesis in cancer therapy-endostatin and its mechanisms of action[J]. Exp Cell Res, 2006,312(5): 594～607.
72．刘颖，周清华，陆艳蓉，等．非小细胞肺癌患者血清中内皮抑素、VEGF水平及其与临床病理生理特征的关系[J]．中国肺癌杂志，2004，7(1)：50～54
73. Lizasa T, Chang H, Suzuki M, et al. Overexpression of collagen ⅹ ⅷis associated with poor outcome and elevated level s of circulating serum endostatin in non-small cell lung cancer [J]. Clin Cancer Res. 2004,10(16):5361-5366
74. Feldman A L, Tamarkin L, Paciotti G F, et al.Serum endostatin levels are elevated and correlate with serum vascular endothelial growth factor levels in patients with stage Ⅳ clear cell renal cancer [J]. Clin Cancer Res, 2000, 6 (12): 4628-4634.
75. Kugler A,Hemmerlein B, Thelen P, et al.Expression of metalloproteinase 2 and 9 and their inhibitors in renal cell carcinoma[J]. J Urol, 1998, 160 (5):1914-1918.
76. Sasaki T, Fukai N,Mann K, et al. Structure,function and tissue forms of the C2terminal globular domain of collagen containing the angiogenesis inhibitor endostatin [J]. EMBO J, 1998,17(15): 4249-4256.
1. Rowhrborm CG, McConnell JD. Campbell's Urology[M]. Philadelphia: W.B. Saunders Company,2002:chapt38,1297～1330.
2. Berry S J, Coffey D S, Walsh P C, et al. The development of human benign prostatic hyperplasia with age[J]. J Urol,1984,132 (3):474～479.
3. Barry M, Roehrborn C. Management of benign prostatic hyperplasia[J]. Annu Rev Med,1997,48: 177～189.
4. Oesterling J E. Benign prostatic hyperplasia: a review of its histogenesis and natural history[J]. Prostate Suppl,1996, 6: 67～73.
5. Berry S J,Coffery D S,Walsh P C,et al.The development of human benign prostatic hyperplasia with age[J].J Urol,1984, 132 (2): 474～479.
6．夏同礼，孔祥田等．我国成人良性前列腺增生的发生与组织形态特征[J]．中华泌尿外科杂志，1993，6：415．
7. Berry S J, Coffey D S, Ewing L L. Effects of aging on prostate growth in beagles[J]. Am J Physiol, 1986,250(6pt2): 1039～1046.
8．顾方六．前列腺增生和前列腺癌在中国发病的初步探讨[J]．中华外科杂志，1993，31：323-326．
9．郭应禄，胡礼泉等．前列腺癌 男科学[M]．北京：人民卫生出版社，2004：1513．
10．郭应禄，李宏军．前列腺炎的流行病学[M]．北京：人民军医出版社，2002：89．
11．夏同礼，孔祥田，宓培，等．我国成人前列腺非特异性炎[J]．中华泌尿外科杂志，1995，16(12)：711～712．
12. Mehik A, Hellstrom P, Lukkarinen O, et al, Epidemiology of prostatitis in Finnish men: a population-based cross-sectional study[J]. BJU Int, 2000, 86(4):443～448.
13. Robertson C Boyle P,Nonis A,et al. International population bascd study of urological conditions: the Urepik study. Ⅱ. Comparative prostatitis data[J]. J Urol, 1999,161(4): 32.
14. Wenninger K, Heiman J R, Rothman I,et al. Sickness impact of chronic nonbacterial prostatitis and its correlates [J]. J Urol, 1996 ,155(3): 965-968.
15. Grayhack J T, Kozlowski J M, Lee C. The pathogenesis of benign prostatic hyperplasia: a proposed hypothesis and critical evaluation[J]. J Urol, 1998,160(6pt2): 2375-2380.
16. Farnsworth W E.Prostate stroma: physiology [J]. Prostate ,1999,38(1): 60-72
17. Cai Y. Benign prostatic hyperplasia is a reawakened process ofpersistent Mullerian duct mesenchyme[J]. BJU Int,2001, 87(3): 177-182.
18. Mc Vary K T, McKenna K E, Lee C. Prostate innervation[J]. Prostate Suppl, 1998,8:2-13.
19. Pennefather J N, Lau W A, Mitchelson F, et al. The autonomic and sensory innervation of the smooth muscle of the prostate gland: a review of pharmacological and histological studies[J]. J Auton Pharmacol,2000,20(4): 193-206.
20. Andersson K E, Lepor H, Wyllie M G.. Prostatic alphal-adrenoceptors and uroselectivity[J]. Prostate ,1997,30(3): 202-215.
21. Hieble J P, Ruffolo R R. The use of alpha-adrenoceptor antagonists in the pharmacological management of benign prostatic hypertrophy: an overview. [J]. Pharmacol Rev Commun, 1996,33(3):145-160.
22 Lepor H, Williford W O, Barry M J, et al. The impact of medical therapy on bother due to symptoms, quality of life and global outcome, and factors predicting response. Veterans Affairs Cooperative Studies Benign Prostatic Hyperplasia Study Group[J]. J Urol, 1998,160(4): 1358-1367.
23. Coffey D S. The molecular biology of the prostate[M]. In H: Lepor, 1993: 28-56
24. Farnsworth W E. Role of estrogen and SHBG in prostate physiology [J]. Prostate, 1996,28(1):17-23.
25. Hudson D L, Guy A T, Fry P, et al. Epithelial cell differentiation pathways in the human prostate. Identification of intermediate phenotypes by keratin expression[J]. J Histochem Cytochem, 2001,49(2):271-278.
26. Risbridger G , Wang H ,Young P,et al. Evidence that epithelial and mesenchymal estrogen receptor-a mediates effects of estrogen on prostatic epithelium[J]. Dev Biol, 2001, 229(2): 432-442.
27. El-Alfy M, Pelletier G, Hermo L S, et al. Unique featuresof the basal cells of human prostate epithelium[J]. Microsc Res Tech,2000,51(5):436-446.
28. Habermann H, Chang W Y, Birch L, et al. Developmental exposure to estrogens alters epithelial cell adhesion and gap junction proteins in the adult rat prostate[J].Endocrinology,2001,142(1):359-369.
29. Kwan P W, Merk F B, Leav I,et al. Estrogen-mediated exocytosis in the glandular epithelium of prostates in castrated and hypophysectomized dogs[J]. Cell Tissue Res ,1982,226(3):689- 693.
30. Aumuller G, Leonhardt M, Janssen M, et al. Neurogenic origin of human prostate endocrinecells[J]. Urology,1999, 53(5):1041-1048.
31. Martin R, Fraile B, Peinado F, et al. Immunohistochemical localization of protein gene product 9.5, ubiquitin, and neuropeptide Yimmunoreactivities in epithelial and neuroendocrine cells from normal and hyperplastic human prostate[J]. J Histochem Cytochem,2000,48(8):1121-1130.
32. Hakanson R, Larsson L I, Sjoberg N O,et al. Amine-producing endocrine-like cells in the epithelium of urethra and prostate of the guinea-pig. A chemical, fluorescence histochemical, and electron microscopic study[J]. Histochemie ,1974, 38(3):259-270.
33. Aumuller G, Renneberg H, Schiemann P J, et al. The role of apocrine releasedproteins in the post-testicular regulation of human sperm function[J]. Adv Exp Med Biol,1997,424:193-219.
34. Kravets F G, Lee J, Singh B, et al. Prostasomes: current concepts[J]. Prostate ,2000, 43(3): 169-174.
35. Saez F, Motta C, Boucher D,et al. Prostasomes inhibit the NADPH oxidase activity of human neutrophils[J]. Mol Hum Reprod,2000, 6(10):883-891.
36. Acosta S, Dizeyi N, Pierzynowski S, et al. Neuroendocrine cells and nerves in the prostate of the guineapig: effects of peripheral denervation and castration[J]. Prostate,2001,46(3): 191-199.
37. Tabassian A R, Nylen E S, Lukacs L, et al. Cholinergic regulation of hamster pulmonary neuroendocrine cell calcitonin[J]. Exp Lung Res, 1990, 16(3):267-277.
38. Simak R, Capodieci P, Cohen D W, et al. Expression of c-kit and kit-ligand in benign and malignant prostatic tissues[J]. Histol Histopathol,2000,15(2): 365-374.
39. Keast J R, Saunders R J. Testosterone has potent, selective effects on the morphology of pelvic autonomic neurons which control the bladder, lower bowel and internal reproductive organs of the male rat[J]. Neuroscience, 1998,85(2): 543-556.
40. Kihara K, Sato K, Ishizaka K, et al. Preservation of ejaculatory function by reconstruction of the canine hypogastric nerve. J Neurosurg 1998,88(4): 726-733.
41. Andersson K E. The concept of uroselectivity. Eur Urol, 1998,33(suppl 2), 7-11.
42. Ruggieri M R, Colton M D, Wang P, et al. Human prostate muscarinic receptor subtypes[J]. J Pharmacol Exp Ther,1995,274(2):976-982.
43. Kostenis E, Zeng FY, Wess J. Structure-function analysis of muscarinic receptors and their associated G proteins[J]. Life Sci,1999,64(6-7):355-362.
44. Onali P, Olianas MC.Bimodal regulation of cyclic AMP by muscarinic receptors. Involvement of multiple G proteins and different forms of adenylyl cyclase[J].life Sci.l995,56(11/12):973-980.
45. Felder cc.Muscarinic acetylcholine receptors:signal transduction through multiple effectors[J].FASEB J,1995,9(8):619-625.
46. Chappie C R, Crowe R, Gilpin S A, et al. The innervation of the human prostate gland—the changesassociated with benign enlargement[J]. J Urol, 1991,146(6): 1637-1644.
47. Hedlund P, Ekstrom P, Larsson B, et al. Heme oxygenase and NO-synthase in the human prostate—relation to adrenergic, cholinergic and peptide-containing nerves[J]. J Auton Nerv Syst,1997,63(3):115-126.
48. Dail W G. Autonomic innervation of male reproductive genitalia.In C. A. Maggi (Ed.), Nervous Control of the Urogenital System[M]. Switzerland: Harwood Academic Publishers, 1993: 69-101
49. Sjostrand N O, Ehren I, Eldh J,et al. NADPH diaphorase in glandular cells and nerves and its relation to acetylcholinesterase- positive nerves in the male reproductive tract of man and guinea-pig[J]. Urol Res, 1998,26(3): 181-188.
50. Dixon J S, Jen P Y,Gosling J A. The distribution of vesicularacetylcholine transporter in the human malegenitourinary organsand its co-localization with neuropeptide Y and nitric oxide synthase[J].Neurourol Urodyn,2000,19(2):185-194.
51. Kaleczyc J, Timmermans J P, Majewski M,et al. Immunohistochemical properties of nerve fibres supplying accessory male genital glands in the pig. A colocalisation study[J]. Histochem Cell Biol,1999,111(3):217-228.
52. Gkonos P J, Guo F, Burnstein K L. Type 1 vasoactive intestinal peptide receptor expression in PC3/AR cells is evidence of prostate epithelial differentiation[J]. Prostate.2000,42(2): 137-144.
53. Reubi J C, Laderach U, Waser B, et al. Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor subtypes in human tumors and their tissues of origin[J]. Cancer Res,2000,60(11): 3105-3112.
54. Lau W A, Pennefather J N, Mitchelson F J. Cholinergic facilitation of neurotransmission to the smooth muscle of the guinea-pig prostate gland[J]. Br J Pharmacol,2000,130(5): 1013-1020.
55. Davis B, Goepel M, Bein S, et al. Lack of neuropeptide Y receptor detection inhuman bladder and prostate[J]. BJU Int,2000,85(7):918-924.
56. Lau W A, Pennefather J N. Muscarinic receptor subtypes in the rat prostate gland[J]. Eur J Pharmacol,1998,343(2-3):151-156.
57. Fernandez J L, Rivera L, Lopez P G, et al. Characterization of the muscarinic receptor mediating contraction of the dog prostate[J]. J Auton Pharmacol, 1998,18(4):205-211.
58. Ellis J, Seidenberg M.. Interactions of alcuronium, TMB-8, and other allosteric ligands with muscarinic acetylcholine receptors: studies with chimeric receptors[J]. Mol Pharmacol,2000,58(6):1451-1460.
59. Pontari M A, Luthin G R, Braverman A S, et al. Characterization of muscarinic cholinergic receptor subtypes in rat prostate[J]. J Recept Signal Transduct Res,1998,18(2-3): 151-166.
60. Hedlund H, Andersson K E, Larsson B. Alpha-adrenoceptors and muscarinic receptors in the isolated human prostate[J]. J Urol,1985,134(6):1291-1298.
61. Caulfield M P. Muscarinic receptors-characterization, couplingand function. Pharmacol Ther,1993,58(3):319-379.
62. Luthin G R, Wang P, Zhou H, et al. Role of ml receptor-G protein coupling in cell proliferation in the prostate. Life Sci ,1997,60(13-14):963-968.
63. Tayebati S K, El-Assouad D, Ricci A,et al. Peripheral blood lymphocytes muscarinic cholinergic receptor subtypes in Alzheimer's disease: a marker of cholinergic dysfunction? [J]. J Neuroimmunol, 2001 ,121(1-2): 126-31
64. Obara K, Arai K, Miyajima N, et al.. Expression of m2 muscarinic acetylcholine receptor mRNA in primary culture of human prostate stromal cells[J]. Urol Res,2000,28(3): 196-200.
65. Mottet N, Costa P, Bali JP. Autonomic nervous system and prostatic physiology. Specific features of the alpha-adrenergic system[J]. Prog Urol,1999,9(1):26-36.
66. Rayford W, Noble M J, Austenfeld M A, et al. Muscarinic cholinergic receptors promote growth of human prostate cancer cells[J]. Prostate ,1997,30 (3):160-166.
67. Wessler I, Kirkpatrick C J, Racke K. The cholinergic 'pitfall': acetylcholine, a universal cell molecule in biological systems, including humans[J]. Clin Exp Pharmacol Physiol, 1999,26(3): 198-205.
68. Eglen R M, Hegde S S, Watson N. Muscarinic receptorsubtypes and smooth muscle function[J]. Pharmacol Rev, 1996,48(4):531-565.
69. Ehlert F J, Ostrom R S, Sawyer G W. Subtypes of muscarinicreceptor in smooth muscle[J]. Life Sci,1997,61(18): 1729-1740.
70. Ehlert F J, Sawyer G W, Esqueda E E. Contractile roles ofM2 and M3 muscarinic receptors in gastrointestinal smooth muscle[J]. LifeSci, 1999, 64(6-7):387-394.
71. Andersson KE. The importance of the cholinergic system in neurourology[J]. Eur Urol, 1998,34( Suppl 1 ):6-9.
72. Todorov L D, Mihaylova-Todorova S T, Bjur R A,. Differential cotransmission in sympathetic nerves: role of frequency of stimulation and prejunctional autoreceptors[J]. J Pharmacol Exp Ther,1999,290(1):241-246.
73. Lau WA, Pennefather JN, Mitchelson FJ. Cholinergic facilitation of neurotransmission to the smooth muscle of the guinea-pig prostate gland[J]. Br J Pharmacol,2000,130(5): 1013-1020
74. Najbar-Kaszkiel A T, Di Iulio J L,et al. Characterisation of excitatory and inhibitory transmitter systems in prostate glands of rats, guinea pigs, rabbits and pigs[J]. Eur J Pharmacol,1997,337(2-3):251-258.
75. Fernandez J L, Rivera L, Lopez P G,et ai. Characterization of the muscarinic receptor mediating contraction of the dog prostate[J]. J Auton Pharmacol, 1998,18(4):205-211
76. Stevens L A, Chappie C R, Chess-Williams R. Human Idiopathic and Neurogenic Overactive Bladders and the Role of M(2) Muscarinic Receptors in Contraction[J]. Eur Urol. 2006 , [Epub ahead of print]
77. Marshall I, Burt R P, Chappie C R. Signal transduction pathways associated with al-adrenoceptor subtypes in cells and tissues including human prostate[J]. Eur Urol, 1999,36 (suppl 1):42-47.
78. McVary K T, Razzaq A, Lee C, et al. Growth of the rat prostate gland is facilitated by the autonomic nervous system[J]. Biol Reprod,1994,51(1): 99-107.
79. Grando S A. Biological functions of keratinocyte cholinergic receptors[J]. J Invest Dermatol Symp Proc ,1997,2(1): 41-48.
80. Klapproth H, Reinheimer T, Metzen J,et al. Non-neuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man[J]. Naunyn Schmiedebergs Arch Pharmacol, 1997,355(4): 515-523.
81. Slater M, Barden J A, Murphy C R. Changes in growth factor expression in the ageing prostate may disrupt epithelial-stromal homeostasis[J]. Histochem J ,2000,32(6):357-364.
82. Prenzel N, Zwick E, Daub H, et al. EGF receptor transactivation by G-proteincoupled receptors requires metalloproteinase cleavage of proHB-EGF[J]. Nature ,1999,402(6764):884-888.
83. Peehl D M, Sellers R G. Induction of smooth muscle cell phenotype in cultured human prostatic stromal cells[J]. Exp Cell Res,1997,232(2):208-215.
84. Yang G, Timme T L, Park S H, et al. Transforming growth factor b1 transduced mouse prostate reconstitutions: I. Induction of neuronal phenotypes[J]. Prostate,1997,33(3):151-156.
85. Dalai R, Djakiew D. Molecular characterization of neurotrophinexpression and the corresponding tropomyosin receptor kinases(trks) in epithelial and stromal cells of the human prostate[J]. Mol Cell Endocrinol ,1997,134(1): 15-22.
86. Pongrac J L, Rylett R J. Molecular mechanisms regulating NGF-mediated enhancement of cholinergic neuronal phenotype: c-fos trans-activation of the choline acetyltransferase gene[J]. J Molec Neurosci, 1998,11(1):79-93.
87. Pflug B R, Dionne C, Kaplan D R,et al. Expression of a trk high affinity nerve growth factor receptor in the human prostate[J]. Endocrinology, 1995, 136(1):262-268.
88. Keast J R, Kepper M E. Differential regulation of trkA and p75 in noradrenergic pelvic autonomic ganglion cells after deafferentation of their cholinergic neighbours[J]. Eur J Neurosci,2001,13(2):211-220.
89. Diaz-Meco M T, Municio M M, Frutos S, The product of par-4, a gene induceduring apoptosis, interacts selectively with the atypical isoforms ofprotein kinase C[J]. Cell,1996,86(5):777-786.
90. Solano R M, Carmena M J, Carrero I, et al. Characterization of vasoactive intestinal peptide/pituitary adenylate cyclase- activating peptide receptors in human benign hyperplastic prostate[J]. Endocrinology, 1996,137(7):2815-2822.
91. Guo Q, Fu W, Xie J, et al. Par-4 is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer's disease[J]. Nat Med, 1998,4(8): 957-962.
92. Guo Q, Xie J, Du H. Par-4 induces cholinergic hypoactivityby suppressing ChAT protein synthesis and inhibiting NGF-inducibility of ChAT activity[J]. Brain Res,2000,874(2):221-232.
93. Lujan M, Paez A, Llanes L, et al. Role of autonomic innervation in rat prostatic structure maintenance: a morphometric analysis[J]. J Urol,1998,160(5): 1919-1923.
94. Doggweiler R, Zermann D H, Ishigooka M,et al. Botox-induced prostatic involution[J]. Prostate ,1998,37(1):44-50.
95. Abbou CC, Salomon L, Chopin D, et al. The current approach to the management of benign hypertrophy of the prostate [J]. Ann Urol,1996,30(6-7):294-301.
96. Keast J R. Effects of testosterone on pelvic autonomic pathways :progress and pitfalls[J]. J Auton Nerv Sys,2000,79(2-3):67-73.
97. Crone J K, Burnett A L, Chamness S L, et al. Neuronal nitric oxide synthase in the canine prostate:aging, sex steroid, and pathology correlations [J]. J Androl,1998,19(3):358-364.
98. Papka R E, Traurig H H, Schemann M,et al. Cholinergic neurons of the pelvic autonomic ganglia and uterus of the female rat: distribution of axons and presence of muscarinic receptors[J]. Cell Tissue Res,1999,296:293-305.
99. Slater M, Barden J A, Murphy C R. Tyrosine kinase A, autonomic and transmitter receptors, but not innervation, are upregulated in the aging rat prostate[J]. Acta Histochem,2000,102(4):427-438.
100. Horsfall D J, Mayne K, Ricciardelli C, et al. Age-related changes in guinea pig prostatic stroma[J]. Lab Invest ,1994,70(5):753-763.
101. Levin R M, Haugaard N, O'Connor L, et al. Obstructive response of human bladder to BPH vs. rabbit bladder response to partial outlet obstruction: a direct comparison[J]. Neurourol Urodyn ,2000,19(5):609- 629.
102. Bayliss M, Wu C, Newgreen D,et al. A quantitative study of atropine-resistant contractile responses in human detrusor smooth muscle, from stable, unstable and obstructed bladders[J]. J Urol,1999,162(5): 1833-1839.
103. Sullivan J, Abrams P. Pharmacological management of incontinence[J]. Eur Urol,1999,36 (suppll): 89-95.
104. Ruffolo R R, Jr, Hieble J P. Adrenoceptor pharmacology: urogenital applications[J]. Eur Urol, 1999,36 (suppl 1):17- 22.
105. Szell E A, Yamamoto T, De Groat W C, et al. Smooth muscle and parasympathetic nerve terminals in the rat urinary bladder have different subtypes of al adrenoceptors[J]. Br J Pharmacol,2000,130(7):1685-1691.
106. Corvin S,Strasser H,Boesch ST, et al. Human rhabdosphincter cell culture:a model for video microscopy of cell contractions[J]. Prostate,2001,47(3): 189-193.
107. Rimmaudo L E,de la Torre E,Sacerdote de Lustig E,et al. Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis[J].Biochem Biophys Res Commun,2005,334(4): 1359-64.
108. Rayford W, Noble M J, Austenfeld M A, et al. Muscarinic cholinergic receptors promote growth of human prostate cancer cells. Prostate[J]. 1997 Feb 15;30(3): 160-166
109. Sydorenko V, Shuba Y, Thebault S. Receptor-coupled, DAG-gated Ca2+-permeable cationic channels in LNCaP human prostate cancer epithelial cells[J]. J Physiol,2003,548(Pt 3):823-836.
110. Blanco M, Robinson M J. Muscarinic M3 acetylcholine receptor immunostaining in paraffin-embedded normal and neoplastic prostatic gland tissue[J]. Ann Diagn Pathol,2004,8(6):333-336.
111. Kester R R, Mooppan U M, Gousse A E,et al. Pharmacological characterization of isolated human prostate[J]. J Urol,2003 ,170(3):1032-1038.
1 Huggins C, Hodges CV. Studies in prostate cancer. I. The effects of castration, of oestrogen, and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res ,1941,1:293-297.
2 Petrylak DP. The current role of chemotherapy in metastatic hormone refractory prostate cancer. Urology, 2005,65 (5 Suppl):3-8.
3 Ganry O. Phytoestrogens and prostate cancer risk. Prev Med,2005,41(1):1-6.
4 Holzbeierlein JM, McIntosh J, Thrasher JB. The role of soy phytoestrogens in prostate cancer. Curr Opin Urol 2005,15(1):17—22.
5 Dalgleish A, Whelan M. Novel immunotherapeutic approaches to prostate cancer. Curr Opin Mol Ther, 2005,7(1):30-34.
6 Tammela T. Endocrine treatment of prostate cancer. J Steroid Biochem Mol Biol, 2004,92(4):287-295.
7 Chang C, Saltzman A, Yeh S, et al.Androgen Receptor: An Overview. Critical Reviews in Eukaryotic Gene Expression, 1995,5(2):97—125.
8 Kyprianou N, English HF, Isaacs JT. Programmed cell death during regression of PC-82 human prostate cancer following androgen ablation. Cancer Res ,1990,50(12):3748-3753.
9 Chen CD, Welsbie DS, Tran C, et al.Molecular determinants of resistance to antiandrogen therapy. Nat Med, 2004,10(1):33-39.
10 Visakorpi T, Hyytinen E, Koivisto P, et al. In vivo amplification of the androgen receptor gene and progression of human prostate cancer. Nat Genet , 1995, 9(4):401-406.
11 Koivisto P, Kononen J, Palmberg C, et al. Androgen receptor gene amplification: A possible molecular mechanism for androgen deprivation therapy failure in prostate cancer. Cancer Res , 1997,57(2):314-319.
12 Eder IE, Culig Z, Ramoner R, et al. Inhibition of LNCaP prostate cancer cells by means of androgen receptor antisense oligonucleotides. Cancer Gene Therapy ,2000,7(7):997-1007.
13 Eder IE, Hoffmann J, Rogatsch H, et al. Inhibition of LNCaP prostate tumor growth in vivo by an antisense oligonucleotide directed against the human androgen receptor. Cancer Gene Therapy, 2002,9(2): 117-125.
14 Ko YJ, Devi GR, London CA, et al.Androgen receptor down-regulation in prostate cancer with phosphorodiamidate morpholino antisense oligomers. J Urol,2004,172(3): 1140-4.
15 Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer. Oncogene, 2003,22(56):9087-9096.
16 Zamore PD, Tuschl T, Sharp PA, et al. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell ,2000,101(1):25-33.
17 Haag P, Bektic J, Bartsch G, et al. Androgen receptor down-regulation by small interference RNA induces cell growth inhibition in androgen sensitive as well as in androgen-independent prostate cancer cells. J Steroid Biochem Mol Biol, 2005,96(3-4):251-258.
18 Liao X, Tang S, Thrasher JB, et al. Small-interfering RNA-induced androgen receptor silencing leads to apoptotic cell death in prostate cancer. Mol Cancer Ther, 2005,4(4):505-515.
19 Soutschek J, Akinc A, Bramlage B, et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature ,2004,432(7014): 173-178.
20 Minamiguchi K, Kawada M, Ohba S, et al.Ectopic expression of the amino-terminal peptide of androgen receptor leads to androgen receptor dysfunction and inhibition of androgen receptor-mediated prostate cancer growth. Mol Cell Endocrinol, 2004,214(1-2): 175-187.
21 Bramlett KS, Dits NF, Sui X, et al. Repression of androgen-regulated gene expression by dominant negative androgen receptors. Mol Cell Endocrinol, 2001,183(1-2): 19-28.
22 Zegarra-Moro OL, Schmidt LJ, Huang H, et al. Disruption of androgen receptor function inhibits proliferation of androgen-refractory prostate cancer cells. Cancer Res, 2002,62(4): 1008-1013.
23 Latham JP, Searle PF, Mautner V, et al. Prostate-specific antigen promoter/enhancer driven gene therapy for prostate cancerxonstruction and testing of a tissue-specific adenovirus vector. Cancer Res, 2000,60(2):334-41.
24 Pang S. Targeting and eradicating cancer cells by a prostate-specific vector carrying the diphtheria toxin A gene. Cancer Gene Ther, 2000,7(7):991-996.
25 Ikegami S, Tadakuma T, Suzuki S, et al. Development of gene therapy using prostate-specific membrane antigen promoter/enhancer with Cre Recombinase/LoxP system for prostate cancer cells under androgen ablation condition. Jpn J Cancer Res ,2002,93(10): 1154-63.
26 Kubo H, Gardner TA,Wada Y, , et al.Phase I dose escalation clinical trial of adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormone-refractory prostate cancer. Hum Gene Ther, 2003,14(3):227-241.
27 Schalken JA, Hessels D, Verhaegh G. New targets for therapy in prostate cancer: differential display code 3 (DD3(PCA3)), a highly prostate cancer-specific gene. Urology ,2003,62(5 Suppl 1):34-43.
28 Farokhzad OC, Jon S, Khademhosseini A, Tran TN, Lavan DA,Langer R. Nanoparticle-aptamer bioconjugates: a new approach for targeting prostate cancer cells. Cancer Res 2004;64:7668-72.
29 Marhefka CA, Gao W, Chung K, et al. Design,synthesis, and biological characterization of metabolically stable selective androgen receptor modulators. J Med Chem, 2004,47(4):993-998.
30 Yin D, Gao W, Kearbey JD, et al. Pharmacodynamics of selective androgen receptor modulators. J Pharmacol Exp Ther, 2003,304(3): 1334-1340.
31 Gao W, Kearbey JD, Nair VA, et al.Comparison of the pharmacological effects of a novel selective androgen receptor modulator, the 5a-reductase inhibitor finasteride,and the antiandrogen hydroxyflutamide in intact rats: New approach for benign hyperplasia. Endocrinology, 2004,145(12):5420-5428.
32 Solit DB, Zheng FF, Drobnjak M, et al. 17-Allylamino-17- demethoxygeldanamycin induces the degradation of androgen receptor and HER-2/neu and inhibits the growth of prostate cancer xenografts. Clin Cancer Res, 2002,8(5):986-993.
33 Vanaja DK, Mitchell SH, Toft DO, et al. Effect of geldanamycin on androgen receptor function and stability. Cell Stress Chaperones ,2002,7(1):55-64.
34 Reed JC. Dysregulation of apoptosis in cancer. J Clin Oncol , 1999, 17(9):2941-53.
35 Raffo AJ, Perlman H, Chen M-W, et al.Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res ,1995,55(19):4438-4445.
36 Yano J, Hirabayashi K, Nakagawa S, et al. Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer. Clin CancerRes, 2004,10(22):7721-7726.
37 Catz SD, Johnson JL. BCL-2 in prostate cancer: a minireview.Apoptosis, 2003,8(1):29-37.
38 Klasa RJ, Gillum AM, Klem RE, et al. Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment. Antisense Nucleic Acid Drug Dev, 2002,12(3): 193-213.
39 Chi KN. Targeting Bcl-2 with oblimersen for patients with hormone refractory prostate cancer. World J Urol ,2005,23(1):33-7.
40 Tolcher AW, Chi K, Kuhn J, et al.A phase II, pharmacokinetic, and biological correlative study of oblimersen sodium and docetaxel in patients with hormone-refractory prostate cancer. Clin Cancer Res ,2005,11(10):3854—61.
41 Morris MJ, Cordon-Cardo C, Kelly WK, et al. Safety and biologic activity of intravenous BCL-2 antisense oligonucleotide (G3139) and taxane chemotherapy in patients with advanced cancer. Appl Immunohistochem Mol Morphol, 2005,13(1):6-13.
42 De Bono JS, Rowinsky EK, Kuhn J, et al. Phase I Pharmacokinetic (PK) and Pharmacodynamic (PD) Trial of bcl-2 antisense (genasense) and docetaxel (D) in hormone refractory prostate cancer. Proceedings of the American Society of Clinical Oncology, 37th Annual Meeting 2001 ;abstract 474.
43 Steinberg J, Oyasu R, Lang S, et al.Intracellular levels of SGP-2 (Clusterin) correlate with tumor grade in prostate cancer. Clin Cancer Res , 1997, 3(10):1707-1711.
44 Pins MR, Fiadjoe JE, Korley F, et al. Clusterin as a possible predictor for biochemical recurrence of prostate cancer following radical prostatectomy with intermediate Gleason scores: a preliminary report. Prostate Cancer Prostatic Dis,2004,7(3):243-248.
45 Gleave M, Miyake H. Use of antisense oligonucleotides targeting the cytoprotective gene, clusterin, to enhance androgen- and chemo-sensitivity in prostate cancer. World J Urol, 2005,23(1):38-46.
46 Springate CM, Jackson JK, Gleave ME, et al. Efficacy of an intratumoral controlled release formulation of clusterin antisense oligonucleotide complexed with chitosan containing paclitaxel or docetaxel in prostate cancer xenograft models. Cancer Chemother Pharmacol, 2005,56(3):239-247.
47 Horiatis D, Wang Q, Pinski J. A new screening system for proliferation-independent anti-cancer agents. Cancer Lett, 2004,210(1):119-24.
48 Tombal B, Denmeade SR, Gillis JM, et al. A supramicromolar elevation of intracellular free calcium ([Ca(2+)](i)) is consistently required to induce the execution phase of apoptosis. Cell Death Differ, 2002,9(5):561-573.
49 Furuya Y, Lundmo P, Short AD, et al. The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin. Cancer Res , 1994, 54(23): 6167-75.
50 Denmeade SR, Jakobsen CM, Janssen S, et al. Prostate-specific antigen-activated thapsigargin prodrug as targeted therapy for prostate cancer. J Natl Cancer Inst, 2003,95(13):990-1000.
51 Denmeade SR, Isaacs JT. The SERCA Pump as a Therapeutic Target:Making a "Smart Bomb" for Prostate Cancer. Cancer Biol Ther, 2005,4(1): 14-22.
52 Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network.Nat Rev Mol Cell Biol, 2001,2(2):127-37.
53 El-Rayes BF, LoRusso PM. Targeting the epidermal growth factor receptor. Br J Cancer 2004;91(3):418-24.
54 Bartlett JM, Brawley D, Grigor K, et al.Type I receptor tyrosine kinases are associated with hormone escape in prostate cancer. J Pathol, 2005,205(4):522-9.
55 Mass RD. The HER receptor family: a rich target for therapeutic development. Int J Radiat Oncol Biol Phys, 2004,58(3):932-940.
56 Lorenzo GD, Bianco R, Tortora G, et al. Involvement of growth factor receptors of the epidermal growth factor receptor family in prostate cancer development and progression to androgen independence. Clin Prostate Cancer, 2003, 2(1):50-57.
57 Zellweger T, Ninck C, Bloch M, et al. Expression patterns of potential therapeutic targets in prostate cancer. Int J Cancer, 2005,113(4):619-628.
58 Signoretti S, Montironi R, Manola J, et al. Her-2-neu expression and progression toward androgen independence in human prostate cancer. J Natl Cancer Inst, 2000,92(23): 1918-1925.
59 Sirotnak FM, She Y, Lee F, rt al. Studies with CWR22 xenografts in nude mice suggest that ZD1839 may have a role in the treatment of both androgen-dependent and androgen-independent human prostate cancer. Clin Cancer Res 2002;8(12):3870-3876.
60 Baselga J, Rischin D, Ranson M, et al. Phase I safety, pharmacokinetic, and pharmacodynamic trial of ZD1839, a selective oral epidermal growth factor receptor tyrosine kinase inhibitor, in patients with five selected solid tumor types. J Clin Oncol, 2002,20(21):4292-302.
61 Lorusso PM. Phase I studies of ZD1839 in patients with common solid tumors. Semin Oncol, 2003,30 (1 Suppl 1):21-29.
62 Canil CM, Moore MJ, Winquist E, et al. Randomized phase II study of two doses of gefitinib in hormone-refractory prostate cancer: a trial of the National Cancer Institute of Canada-Clinical Trials Group. J Clin Oncol . 2005. 23(3):455-4560.
63 Solit DB, She Y, Lobo J, et al. Pulsatile administration of the epidermal growth factor receptor inhibitor gefitinib is significantly more effective than continuous dosing for sensitizing tumors to paclitaxel. Clin Cancer Res, 2005, 11(5):1983—1989.
64 Von Pawel J. Gefitinib (Iressa, ZD1839): a novel targeted approach for the treatment of solid tumors. Bull Cancer, 2004,91 (5):E70-6.
65 Klapper LN, Glathe S, Vaisman N, et al. The ErbB-2/HER2 oncoprotein of human carcinomas may function solely as a shared coreceptor for multiple stroma-derived growth factors. Proc Natl Acad Sci USA, 1999, 96(9): 4995-5000.
66 Hackel PO, Zwick E, Prenzel N, et al. Epidermal growth factor receptors: critical mediators of multiple receptor pathways. Curr Opin Cell Biol, 1999,11(2): 184-9.
67 Klapper LN, Kirschbaum MH, Sela M, et al. Biochemical and clinical implications of the ErbB/HER signaling network of growth factor receptors. Adv Cancer Res, 2000,77:25-79.
68 Baselga J. A new anti-ErbB2 strategy in the treatment of cancer:prevention of ligand-dependent ErbB2 receptor heterodimerization. Cancer Cell, 2002,2(2):93-95.
69 Agus DB, Akita RW, Fox WD, et al. Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell, 2002,2(2): 127-137.
70 Mendoza N, Phillips GL, Silva J, et al. Inhibition of ligand-mediated HER2 activation in androgen-independent prostate cancer. Cancer Res ,2002, 62(19):5485-5488.
71 Craft N, Shostak Y, Carey M, et al. A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat Med 1999;5(3):280-285.
72 Lara Jr PN, Chee KG, Longmate J, et al. Trastuzumab plus docetaxel in HER-2/neu-positive prostate carcinoma: final results from the California Cancer Consortium Screening and Phase II Trial. Cancer ,2004,100(10):2125-2131.
73 Chung LW, Baseman A, Assikis V, et al. Molecular insights into prostate cancer progression: the missing link of tumor microenvironment.J Urol , 2005, 173(1): 10-20.
74 van Moorselaar RJ, Voest EE. Angiogenesis in prostate cancer: its role in disease progression and possible therapeutic approaches. MolCell Endocrinol 2002;197(l-2):239-50.
75 Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell ,1991,64(2):327-36.
76 Folkman J, Shing Y, Angiogenesis. J Biol Chem 1992;267(16): 10931-10934.
77 Jiang WG, Puntis MC, Hallett MB. Molecular and cellular basis of cancer invasion and metastasis: implications for treatment. Br J Surg 1994;81(11): 1576-90.
78 Weidner N, Carroll PR, Flax J,et al. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma.Am J Pathol ,1993,143(2):401-9.
79 Jackson MW, Roberts JS, Heckford SE, et al. A potential autocrine role for vascular endothelial growth factor in prostate cancer. Cancer Res, 2002,62(3):854-9.
80 Doll JA, Reiher FK, Crawford SE, et al. Thrombospondin-1, vascular endothelial growth factor and fibroblast growth factor-2 are key functional regulators of angiogenesis in the prostate. Prostate 2001;49(4):293-305.
81 Chan LW, Moses MA, Goley E, et al. Urinary VEGF and MMP levels as predictive markers of 1 -year progression-free survival in cancer patients treated with radiation therapy: a longitudinal study of protein kinetics throughout tumor progression and therapy. J Clin Oncol, 2004,22(3):499-506.
82 Melnyk O, Zimmerman M, Kim KJ, et al. Neutralizing antivascular endothelial growth factor antibody inhibits further growth of established prostate cancer and metastases in a pre-clinical model. J Urol ,1999,161(3):960-3.
83 Petrylak DP. Future directions in the treatment of androgen-independent prostate cancer. Urology ,2005,65 (6 Suppl):8-12.
84 Figg WD, Kruger EA, Price DK, et al. Inhibition of angiogenesis: treatment options for patients with metastatic prostate cancer. Invest New Drugs ,2002,20(2): 183-94.
85 Nicholson B, Guiding K, Conaway M, et al.Combination antiangiogenic and androgen deprivation therapy for prostate cancer: a promising therapeutic approach. Clin Cancer Res, 2004,10(24):8728-8734.
86 Pettit GR, Rhodes MR, Herald DL, et al. Antineoplastic agents. 445. Synthesis and evaluation of structural modifications of (Z)- and (E)-combretastatin A-41. J Med Chem, 2005,48(12):4087-99.
87 Leblanc R, Dickson J, Brown T, et al. Synthesis and cytotoxicity of epoxide and pyrazole analogs of the combretastatins. Bioorg Med Chem, 2005, 13(21):6025-6034
88 Siemann DW, Bibby MC, Dark GG, et al. Differentiation and definition of vascular-targeted therapies.Clin Cancer Res ,2005,11(2 Pt 1):416—20.
89 Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature ,1988,322(6163):411-5.
90 Battistini B, Chailler P, D'Orleans-Juste P, et al. Growth regulatory properties of endothelins. Peptides ,1993.14(2):385-399.
91 Vacca F, Bagnato A, Catt KJ, et al. Transactivation of the epidermal growth factor receptor in endothelin-1-induced mitogenic signaling in human ovarian carcinoma cells. Cancer Res ,2000,60(18):5310-7.
92 Nelson JB. Endothelin receptor antagonists. World J Urol ,2005,23(1):19-27.
93 Jimeno A, Carducci M. Atrasentan: a novel and rationally designed therapeutic alternative in the management of cancer. Expert Rev Anticancer Ther, 2005,5(3):419-27.
94 Stewart DA, Cooper CR, Sikes RA. Changes in extracellular matrix (ECM) and ECM-associated proteins in the metastatic progression of prostate cancer. Reprod Biol Endocrinol 2004;2:2.
95 Kleiner DE, Stetler-Stevenson WG. Matrix metalloproteinases and metastasis. Cancer Chemother Pharmacol 1999;43 Suppl:S42-51.
96 Trudel D, Fradet Y, Meyer F,et al. Significance of MMP-2 expression in prostate cancer: an immunohistochemical study. Cancer Res, 2003,63(23): 8511-5.
97 Zhang L, Shi J, Feng J, et al. Type IV collagenase (matrix metalloproteinase-2 and -9) in prostate cancer.Prostate Cancer Prostatic Dis, 2004,7(4):327-32.
98 Knox JD, Bretton L, Lynch T, et al. Synthetic matrix metalloproteinase inhibitor, BB-94, inhibits the invasion of neoplastic human prostate cells in a mouse model. Prostate, 1998,35(4):248-54.
99 Somlyo AV, Phelps C, Dipierro C, et al. Rho kinase and matrix metalloproteinase inhibitors cooperate to inhibit angiogenesis and growth of human prostate cancer xenotransplants.Faseb J 2003 ;17(2):223-34.
100 Rosenbaum E, Zahurak M, Sinibaldi V, et al. Marimastat in the treatment of patients with biochemically relapsed prostate cancer: A prospective randomized, double-blind,phase I/II trial. Clin Cancer Res ,2005,11(12):4437-43.
101 Coussens LM, Fingleton B, Matrisian LM. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science, 2002,295(5564):2387-92.
102 Shamsuddin AM, Vucenik I, Cole KE. IP6: a novel anti-cancer agent.Life Sci, 1997,61(4):343-54.
103 Agarwal C, Dhanalakshmi S, Singh RP, et al. Inositol hexaphosphate inhibits growth and induces Gl arrest and apoptotic death of androgen-dependent human prostate carcinoma LNCaP cells.Neoplasia 2004;6(5):646-59.
104 Singh RP, Sharma G, Mallikarjuna GU, et al. In vivo suppression of hormone-refractory prostate cancer growth by inositol hexaphosphate: induction of insulin-like growth factor binding protein-3 and inhibition of vascular endothelial growth factor. Clin Cancer Res, 2004,10(1 Pt 1)::244-250.
105 Adams J, Palombella VJ, Sausville EA, et al. Proteasome inhibitors: A novel class of potent and effective antitumor agents. Cancer Res, 1999,59(11): 2615-2622.
106 Herrmann JL, Briones Jr F, Brisbay S, et al.Prostate carcinoma cell death resulting from inhibition of proteasome activity is independent of functional bcl-2 and p53. Oncogene ,1998,17(22):2889-99.
107 Williams SA, McConkey DJ. The proteasome inhibitor bortezomib stabilizes a novel active form of p53 in human LNCaP-Pro5 prostate cancer cells. Cancer Res, 2003,63(21):7338-44.
108 Williams SA, Pettaway C, Song R, et al. Differential effects of the proteasome inhibitor bortezomib on apoptosis and angiogenesis in human prostate tumor xenografts. Mol Cancer Ther, 2003,2(9):835-43.
109 Papandreou CN, Daliani DD, Nix D, et al. Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer. J Clin Oncol 2004;22(11):2108—21.
110 Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell, 1994,79(1): 13-21.
111 Price N, Dreicer R. Phase I/II trial of bortezomib plus docetaxel in patients with advanced androgen-independent prostate cancer. Clin Prostate Cancer 2004;3(3): 141-3.
112 Hidalgo M, Rowinsky EK. The rapamycin-sensitive signal transduction pathway as a target for cancer therapy. Oncogene 2000;19(56):6680-6686.
113 Wu L, Birle DC, Tannock IF. Effects of the mammalian target of rapamycin inhibitor CCI-779 used alone or with chemotherapy on human prostate cancer cells and xenografts. Cancer Res, 2005,65(7):2825-2831.
114 Iversen PL, Arora V, Acker AJ, et al. Efficacy of antisense morpholino oligomer targeted to c-myc in prostate cancer xenograft murine model and a Phase I safety study in humans. Clin Cancer Res, 2003,9(7):2510-9.
115 Devi GR, Beer TM, Corless CL, et al. In vivo bioavailability and pharmacokinetics of a c-MYC antisense phosphorodiamidate morpholino oligomer, AVI-4126, in solid tumors. Clin Cancer Res ,2005,11(10):3930-3938.
116 Lee Y, Vassilakos A, Feng N, et al.GTI-2040, an antisense agent targeting the small subunit component(R2) of human ribonucleotide reductase, shows potent antitumor activity against a variety of tumors. Cancer Res, 2003,63(11): 2802-11.
1 1 Pennefather J N, Lau WA, Mitchelson F,et al. The autonomic and sensory innervation of the smooth muscle of the prostate gland: a review of pharmacological and histological studies. J AutonPharmaco,2000, 20(4): 193-206.
2 Ruggieri MR, Colton MD, Wang P, et al.Human prostate muscarinic receptor subtypes. J Pharmacol Exp Ther ,1995,274 (2) : 976-982.
3 Batra S, Christensson PI,Hartley-Asp B. Characterization of muscarinic cholinergic receptors in membrane preparations from rat prostatic adenocarcinoma. Prostate, 1990,17(4): 261-268.
4 of smooth muscle cells from human prostate. J Urol, 1994,152 (6) : 2173- 2177.
5 Obara K, Arai K, Miyajima Ret al..Expression of m2 muscarinic acetylcholine receptor mRNA in primary culture of human prostate stromal cells. Urol Res, 2000,28(3): 196-200.
6 Fernandez JL,Rivera L,Lopez PG,Recio P,et al. Characterization of the muscarinic receptor mediating contraction of the dog prostate. J Auton Pharmacol, 1998,18(4):205-11.
7 Cheng K, Chen Y , Zimniak P,et al. Functional interaction of lithocholic acid conjugates with M3 muscarinic receptors on a human colon cancer cell line. Biochimica et Biophysica Acta , 2002,1588 (1) : 48-55.
8 Rimmaudo LE,de la Torre E,Sacerdote de Lustiq E,et ai.Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis. Biochem Biophys Res Commun,2005,334(4): 1359-64.
9 Luthin G R, Wang P, Zhou H, et al. Role of ml receptor-G protein coupling in cell proliferation in the prostate. Life Sci, 1997,60(13-14): 963- 968.
10 Fiszman G , Cattaneo V , Torre, E, et al. Muscarinic receptors autoantibodies purified from mammary adenocarcinoma-bearing mice sera stimulate tumor progression. International Immunopharmacology, 2006,6( 8): 1323-1330.
11 Umeda E, Aramaki Y. Mori T.et al. Muscarinic receptor subtypes modulate the release of [3H]-noradrenaline in rat spinal cord slices. Brain Research Bulletin. 2006,70 (1) :99-102.
12 Blanco M,Robinson MJ.Muscarinic M3 Acetylcholine Receptor Immunostaining in Paraffin-Embedded Normal and Neoplastic Prostatic Gland Tissue. Annals of Diagnostic Pathologly, 2004,8(6):333-336.
1 Gasparini G,Bevilacqua P ,Bonoldi E , et al . Predictive and prognosis markers in a series of patient s with head and neck squamous cell invasive carcinoma treated with concurrent chemoradiation therapy [J ] . Clin Can Res , 1995 , 1(1): 1375-1383.
2 Hanahan D ,Folkman J . Patterns and emerging mechanism of the angiogenic switch during tumorigenesis [J ] . Cell ,1996 ,86 (3) : 353-364.
3 Kaushal V ,Mukunyadzi P ,Dennis RA , et al . Stage2specific characterization of the vascular endothelial growth factor axis in prostate cancer : expression of lymphangiogenic markers is associated with advanced2stage disease[J ] . Clin Cancer Res ,2005 ,11 (2Pt 1) :584-593.
4 Latil A ,Bieche I , Pesche S , et al . VEGF overexpression in clinically localized prostate tumors and neuropilin21 overexpression in metastatic forms[J ] . Int J Cancer ,2000 ,89 (2) : 1672171.
5 Feldman A L, Tamarkin L, Paciotti G F, et al.Serum endostatin levels are elevated and correlate with serum vascular endothelial growth factor levels in patients with stage IV clear cell renal cancer [J]. Clin Cancer Res, 2000, 6 (12) : 4628- 4634.
6 Kugler A,Hemmerlein B, Thelen P, et al.Expression of metalloproteinase 2 and 9 and their inhibitors in renal cell carcinoma[J]. J Urol, 1998, 160 (5) .1914- 1918.
7 Sasaki T , Fukai N ,Mann K, et al. Structure, function and tissue forms of the C2terminal globular domain of collagen containing the angiogenesis inhibitor endostatin [ J ] . EMBO J , 1998 , 17(15): 4249-4256.
8 Cheng K, Chen Y , Zimniak P,et al. Functional interaction of lithocholic acid conjugates with M3 muscarinic receptors on a human colon cancer cell line. Biochimica et Biophysica Acta, 2002,1588 (1) : 48-55.
9 Fiszman G , Cattaneo V , Torre, E, et al. Muscarinic receptors autoantibodies purified from mammary adenocarcinoma-bearing mice sera stimulate tumor progression. International Immunopharmacology, 2006,6( 8): 1323-1330.
10 Umeda E, Aramaki Y, Mori T,et al. Muscarinic receptor subtypes modulate the releaseof [3H]-noradrenaline in rat spinal cord slices. Brain Research Bulletin, 2006: 70 (1) ,99-102.
2. Dail WG: Autonomic innervation of male reproductive genitalia.In Maggi CA (ed): "Nervous Control of the Urogenital System," Switzerland: Harwood Academic Publishers, 1993:69-101.
3. J .S. Dixon, P.Y.P. Jen, J.A. Gosling, The distribution of vesicular acetylcholine transporter in human male genitourinary organs and its co-localization with neuropeptide Y and nitric oxide synthase, established. Neurourol. Urodyn. 2000:19 185-194.
4. Irving Nadelhaft C holinergic axons in the rat prostate and neurons in the pelvic ganglion Brain Res 2003: 989 52-57
5. Chappie, C. R., Crowe, R., Gilpin, S. A., Gosling, J., & Burnstock, G. The innervation of the human prostate gland—the changes associated with benign enlargement. J Urol 1991:146, 1637- 1644.
6. Gosling JA: Autonomic innervation of the prostate. In Hinman F Jr (ed): "Benign Prostatic Hypertrophy," New York: Springer- Verlag, 1983:349-360.
7. Wang J-M, McKenna KE, Lee C: Determination of prostatic secretion in rats: Effect of neurotransmitters and testosterone. Prostate 1991;18:289-301.
8. Rimmaudo LE,de la Torre E,Sacerdote de Lustiq E,et ai.Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis. Biochem Biophys Res Commun,2005,334(4): 1359-64.
9. Cheng K, Chen Y , Zimniak P,et al. Functional interaction of lithocholic acid conjugates with M3 muscarinic receptors on a human colon cancer cell line. Biochimica et Biophysica Acta, 2002,1588 (1) : 48- 55.)
10. Rimmaudo L E,de la Torre E,Sacerdote de Lustig E,et al. Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis[J].Biochem Biophys Res Commun,2005,334(4): 1359-64.
11. Folkman J . What is the evidence that tumors are angiogenesis dependent. J Natl Cancer Inst, 1990 ;82 (1) :4-6
12. 赵利枝,杨日芳,汪海. 内皮细胞与血管形成. 中国药理学通报,2002;18(4):361-4
13. Ferrer FA ,Miller LJ ,Andrawis RI ,et al. J Urol ,1997 ; 157 :2329-2333
14. Freeman MR ,Schneck FX,Gagnon ML ,et al . Cancer Res ,1995 ;55 :4140-4145
15. Chen HJ, Treweeke AT ,YQ Ke ,et al .Br J Cancer ,2000 ;82 :1694-1701
16. Schuch G, Kisker O, Atala A, et al. Pancreatic tumor growth is regulated by the balance between positive and negative modulators of angiogenesis [J]. Angiogenesis, 2002, 5(3): 181-190.
17. Robak E, Wozniacka A, Sysa-Jedrzejowska A, et al. Circulating angiogenesis inhibitor enodostatin and positive endothelial growth regulators in patients with systemic lupus erythematosus [J]. Lupus, 2002, 11(6):348-355.
18. Chappie C R, Crowe R, Gilpin S A, et al. The innervation of the human prostate gland—the changesassociated with benign enlargement[J]. J Urol, 1991,146(6): 1637-1644.
19. Hedlund P, Ekstrom P, Larsson B, et al. Heme oxygenase and NO-synthase in the human prostate—relation to adrenergic, cholinergic and peptide-containing nerves[J]. J Auton Nerv Syst, 1997,63(3): 115-126.
20. Dail W G. Autonomic innervation of male reproductive genitalia.In C. A. Maggi (Ed.), Nervous Control of the Urogenital System[M]. Switzerland: Harwood Academic Publishers, 1993,69-101
21. Sjostrand N O, Ehren I, Eldh J,et al. NADPH diaphorase in glandular cells and nerves and its relation to acetylcholinesterase- positive nerves in the male reproductive tract of man and guinea-pig[J]. Urol Res,1998,26(3):181-188.
22. Dixon J S, Jen P Y,Gosling J A. The distribution of vesicularacetylcholine transporter in the human malegenitourinary organsand its co-localization with neuropeptide Y and nitric oxide synthase[J].Neurourol Urodyn,2000,19(2): 185-194.
23. Pontari M A, Luthin G R, Braverman A S, et al. Characterization of muscarinic cholinergic receptor subtypes in rat prostate[J]. J Recept Signal Transduct Res,1998,18(2-3):151-166.
24. Hedlund H, Andersson K E, Larsson B. Alpha-adrenoceptors and muscarinic receptors in the isolated human prostate[J]. J Urol,1985,134(6):1291-1298.
25. Caulfield M P. Muscarinic receptors-characterization, couplingand function[J]. Pharmacol Ther,1993,58(3):319-379.
26. Luthin G R, Wang P, Zhou H, et al. Role of ml receptor-G protein coupling in cell proliferation in the prostate[J]. Life Sci ,1997,60(13-14):963-968.
27. Braverman A S ,Tallarida RJ,Ruggieri M R. Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction [J]. Am J Physiol Regul Integr Comp Physiol. 2002 ,283(3):R663-668.
28. Tayebati S K, El-Assouad D, Ricci A,et al. Immunochemical and immunocytochemical characterization of cholinergic markers in human peripheral blood lymphocytes[J]. J Neuroimmunol. 2002,132(l-2):147-155.
29. Ruggieri M R, Colton M D, Wang P, et al. Human prostate muscarinic receptor subtypes[J]. J Pharmacol Exp Ther,1995,274(2):976-982.
30. Fernandez J L, Rivera L, Lopez P G, et al. Characterization of the muscarinic receptor mediating contraction of the dog prostate[J]. J Auton Pharmacol, 1998, 18(4):205-211.
31.Eglen R M, Hegde S S, Watson N. Muscarinic receptorsubtypes and smooth muscle function[J]. Pharmacol Rev,1996,48(4):531-565.
32. Ehlert F J, Ostrom R S, Sawyer G W. Subtypes of muscarinicreceptor in smooth muscle[J]. Life Sci,1997,61(18): 1729-1740.
33. Ehlert F J, Sawyer G W, Esqueda E E. Contractile role ofM2 and M3 muscarinic receptors in gastrointestinal smooth muscle[J]. LifeSci,1999,64(6-7):387-394.
34. Lau W A, Pennefather J N. Muscarinic receptor subtypes in the rat prostate gland[J]. Eur J Pharmacol.1998,343(2-3): 151-156.
35. Obara K, Arai K, Miyajima N, et al. Expression of m2 muscarinic acetylcholine receptor mRNA in primary culture of human prostate stromal cells[J]. Urol Res,2000,28(3): 196-200.
36. Mottet N, Costa P, Bali JP. Autonomic nervous system and prostatic physiology. Specific features of the alpha-adrenergic system[J]. Prog Urol, 1999,9(1):26～36.
37．赵升田 袁明振 王洪伟等．M受体亚型在正常成人前列腺组织中的分布．山东大学学报，2006，44(3)：287-290
38. Rayford W, Noble M J, Austenfeld M A, et al. Muscarinic cholinergic receptors promote growth of human prostate cancer cells[J]. Prostate, 1997,30(3): 160～166.
39. Wessler I, Kirkpatrick C J, Racke K. The cholinergic 'pitfall': acetylcholine, a universal cell molecule in biological systems, including humans[J]. Clin Exp Pharmacol Physiol, 1999,26(3): 198～205.
40. Rayford W, Noble M J, Austenfeld M A, et al. Muscarinic cholinergic receptors promote growth of human prostate cancer cells. Prostate[J]. 1997,30(3): 160～166
41. Sydorenko V, Shuba Y, Thebault S. Receptor-coupled, DAG-gated Ca2+-permeable cationic channels in LNCaP human prostate cancer epithelial cells[J]. J Physiol,2003,548(Pt 3):823～836.
42. Blanco M, Robinson M J. Muscarinic M3 acetytcholine receptor immunostaining in paraffin-embedded normal and neoplastic prostatic gland tissue[J]. Ann Diagn Pathol,2004,8(6):333～336.
43. Borovikova L V, Ivanova S, Zhang M, et al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin [J]. Nature, 2000,405 (6785): 458-462.
44. Kendall M D, al-Shawaf A A. Innervation of rat thymus gland [J]. Brain Behav Immun, 1991, 5(1): 9-28.
45. Gushchin G V, Jakovleva E E, Kataeva G V. Muscarinic cholinergic receptors of rat lymphocytes: effect of antigen stimulation and local brain lesion [J] Neuroimmuno2 modulation, 1994,1 (4): 2592264.
46. Kuo Y, Lucero L, Michaels J, et al. Differential expression of nicotinic acetylcholine receptor subunits in fetal and neonatal mouse thymus [J]. J Neuroimmunol,2002,130(1-2): 140-154.
47. Fujino H, Uehara T, Murayama T, et al. Extracellular signal regulated protein kinase and c2jun N2terminal kinase are involved in ml musearinic receptor2enhanced interleukin22 production pathway in J urkat cells[J]. Biol Pharm B ull, 2000,23(10): 1198-1205.
48．姜建兰，邱一华，彭聿平．乙酰胆碱对自然杀伤细胞活性的影响．中国应用生理学杂志，2005，21(3)，330-332
49. Ferrara N,Houck K,Jakeman L,et al. The vascular endothelial growth factor family of polypeptides [J]. J Cell Biochem, 1991,47(3):211-218
50. Waltenberger J,Welsh LC,Claesson-Siegbahn A,et al. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor[J]. The J Biol Chem,1994,269(43):26988～26995
51. Ferrer FA,Miller LJ, Lindoquist R,et al. Expression of vascular endothelial growth factor receptors in human prostate cancer[J]. Urology,1999,54 (3):567～572
52. Ergun S,Kilic N,Fiedler W,et al. Vascular endothelial growth factor and its receptors in normal human testicular tissue[J]. Mol Cell Endocrinol,1997,131(1):9～20
53. Walther MM,Figg WD,Lineham WM. Intravesical suramin: a novel agent for the treatment of superficial transitional-cell carcinoma of the bladder[J]. World J Urol,1996,14 (suppl1):S8-11
54. Melnyk O,Zimmerman M,Kim KJ,et al. Neutralizing anti-vascular endothelial growth factor antibody inhibits further growth of established prostate cancer and metastases in a pre-clinical model[J]. J Urol, 1999,161 (3):960-963
55. Connolly JM,Rose DP. Angiogenesis in two human prostate cancer cell lines with differing metastatic potential when growing as solid tumors in nude mice[J]. J Urol, 1998,160(3pt1):932～936
56. Hahn D,Simak R,Steiner GE,et al. Expression of the VEGF-receptor Flt-1 in benign, premalignant and malignant prostate tissues[J]. J Urol, 2000,164(2):506～510
57. Latil A,Bieche I,Pesche S,et al. VEGF overexpression in clinically localized prostate tumors and neuropilin-1 overexpression in metastatic forms[J]. Int J Cancer,2000,89(2): 167～171
58. Gaqnon ML,Bielenberg DR, Gechtman Z, et al. Identification of a natural soluble neuropilin-1 that binds vascular endothelial growth factor: In vivo expression and antitumor activity[J]. Proc Natl Acad Sci USA , 2000 , 97(6) :2573-2578
59. Duque JLF ,Loughlin KR ,Adam RM,et al . Plasma levels of vascular endothelial growth factor are increased in patients with metastatic prostate cancer[J]. Urol ,1999 ,54(3) :523- 527
60. Mazzucchelli R ,Montironi R ,Santinelli A ,et al . Vascular endothelial growth factor expression and capillary architecture in high-grade PIN and prostate cancer in untreated and androgen-ablated patients[J]. Prostate ,2000,45(1) :72—79
61. Haggstrom S ,Bergh A ,Damber JE ,et al . Vascular endothelial growth factor content in metastasizing and nonmetastasizing Dunning prostatic adenocarcinoma[J]. Prostate ,2000,45(1) :42-50
62. Liu XH ,Kirschenbaum A ,Yao S ,et al . Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo[J]. J Urol ,2000 ,164(3 Pt l):3820-3825
63. Gaddipati JP ,Mani H ,Shefali ,et al . Inhibition of growth and regulation of IGFs and VEGF in human prostate cancer cell lines by shikonin analogue 93/637 (SA) [J]. Anticancer Res ,2000 ,20(4) :2547-2552
64. Viglietto G,Romano A ,Maglione D ,et al. Neovascularization in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not the placenta-derived growth factor. Oncogene[J] ,1996 ,13(3) :577-587
65. O'Reilly MS ,Boehm T ,Shing Y,et al . Endostantin :an endonous inhibitor of angiogenesis and tumor growt h [ J ]. Cell, 1997 , 88(2) :227 - 285
66. Miosge N , Sasaki T , Timpl R , et al . Angiogenesis inhabitorendostantin is a distinct component of elastic fibers in vessel walls[J ] . FASEB J ,1999 ,13 (13):1743- 1750
67. Sasaki T ,Fukai N ,Mann K,et al. St ruct ure ,function and tissue forms of t he C -terminal globular domain of collegen X VIII containning the angiogenesis inhabitor endostantin [ J ]. EMBO J , 1998 ,17 (15) :4249 - 4256
68. Chen W, Fu J, Liu Q, Ruan C, Xiao S. Retroviral endostatin gene transfer inhibits human colon cancer cell growth in vivo[J]. Chin Med J (Eng1), 2003, 116(10): 1582～1584.
69. Mi J, Sarraf-Yazdi S, Zhang X, Cao Y, Dewhirst MW,Kontos CD, Li CY, Clary BM. A comparison of antiangiogenic therapies for the prevention of liver metastases[J]. J Surg Res, 2006, 131 (1): 97～104.
70．骆成玉，赵丹宁，李世拥，杨晓明，梁龙，祝学光．内皮抑素对结肠癌肝转移的干预作用[J]．中华外科杂志，2001，39(3)：335～337．
71. Folkman J. Antiangiogenesis in cancer therapy-endostatin and its mechanisms of action[J]. Exp Cell Res, 2006,312(5): 594～607.
72．刘颖，周清华，陆艳蓉，等．非小细胞肺癌患者血清中内皮抑素、VEGF水平及其与临床病理生理特征的关系[J]．中国肺癌杂志，2004，7(1)：50～54
73. Lizasa T, Chang H, Suzuki M, et al. Overexpression of collagen ⅹ ⅷis associated with poor outcome and elevated level s of circulating serum endostatin in non-small cell lung cancer [J]. Clin Cancer Res. 2004,10(16):5361-5366
74. Feldman A L, Tamarkin L, Paciotti G F, et al.Serum endostatin levels are elevated and correlate with serum vascular endothelial growth factor levels in patients with stage Ⅳ clear cell renal cancer [J]. Clin Cancer Res, 2000, 6 (12): 4628-4634.
75. Kugler A,Hemmerlein B, Thelen P, et al.Expression of metalloproteinase 2 and 9 and their inhibitors in renal cell carcinoma[J]. J Urol, 1998, 160 (5):1914-1918.
76. Sasaki T, Fukai N,Mann K, et al. Structure,function and tissue forms of the C2terminal globular domain of collagen containing the angiogenesis inhibitor endostatin [J]. EMBO J, 1998,17(15): 4249-4256.
1. Rowhrborm CG, McConnell JD. Campbell's Urology[M]. Philadelphia: W.B. Saunders Company,2002:chapt38,1297～1330.
2. Berry S J, Coffey D S, Walsh P C, et al. The development of human benign prostatic hyperplasia with age[J]. J Urol,1984,132 (3):474～479.
3. Barry M, Roehrborn C. Management of benign prostatic hyperplasia[J]. Annu Rev Med,1997,48: 177～189.
4. Oesterling J E. Benign prostatic hyperplasia: a review of its histogenesis and natural history[J]. Prostate Suppl,1996, 6: 67～73.
5. Berry S J,Coffery D S,Walsh P C,et al.The development of human benign prostatic hyperplasia with age[J].J Urol,1984, 132 (2): 474～479.
6．夏同礼，孔祥田等．我国成人良性前列腺增生的发生与组织形态特征[J]．中华泌尿外科杂志，1993，6：415．
7. Berry S J, Coffey D S, Ewing L L. Effects of aging on prostate growth in beagles[J]. Am J Physiol, 1986,250(6pt2): 1039～1046.
8．顾方六．前列腺增生和前列腺癌在中国发病的初步探讨[J]．中华外科杂志，1993，31：323-326．
9．郭应禄，胡礼泉等．前列腺癌 男科学[M]．北京：人民卫生出版社，2004：1513．
10．郭应禄，李宏军．前列腺炎的流行病学[M]．北京：人民军医出版社，2002：89．
11．夏同礼，孔祥田，宓培，等．我国成人前列腺非特异性炎[J]．中华泌尿外科杂志，1995，16(12)：711～712．
12. Mehik A, Hellstrom P, Lukkarinen O, et al, Epidemiology of prostatitis in Finnish men: a population-based cross-sectional study[J]. BJU Int, 2000, 86(4):443～448.
13. Robertson C Boyle P,Nonis A,et al. International population bascd study of urological conditions: the Urepik study. Ⅱ. Comparative prostatitis data[J]. J Urol, 1999,161(4): 32.
14. Wenninger K, Heiman J R, Rothman I,et al. Sickness impact of chronic nonbacterial prostatitis and its correlates [J]. J Urol, 1996 ,155(3): 965-968.
15. Grayhack J T, Kozlowski J M, Lee C. The pathogenesis of benign prostatic hyperplasia: a proposed hypothesis and critical evaluation[J]. J Urol, 1998,160(6pt2): 2375-2380.
16. Farnsworth W E.Prostate stroma: physiology [J]. Prostate ,1999,38(1): 60-72
17. Cai Y. Benign prostatic hyperplasia is a reawakened process ofpersistent Mullerian duct mesenchyme[J]. BJU Int,2001, 87(3): 177-182.
18. Mc Vary K T, McKenna K E, Lee C. Prostate innervation[J]. Prostate Suppl, 1998,8:2-13.
19. Pennefather J N, Lau W A, Mitchelson F, et al. The autonomic and sensory innervation of the smooth muscle of the prostate gland: a review of pharmacological and histological studies[J]. J Auton Pharmacol,2000,20(4): 193-206.
20. Andersson K E, Lepor H, Wyllie M G.. Prostatic alphal-adrenoceptors and uroselectivity[J]. Prostate ,1997,30(3): 202-215.
21. Hieble J P, Ruffolo R R. The use of alpha-adrenoceptor antagonists in the pharmacological management of benign prostatic hypertrophy: an overview. [J]. Pharmacol Rev Commun, 1996,33(3):145-160.
22 Lepor H, Williford W O, Barry M J, et al. The impact of medical therapy on bother due to symptoms, quality of life and global outcome, and factors predicting response. Veterans Affairs Cooperative Studies Benign Prostatic Hyperplasia Study Group[J]. J Urol, 1998,160(4): 1358-1367.
23. Coffey D S. The molecular biology of the prostate[M]. In H: Lepor, 1993: 28-56
24. Farnsworth W E. Role of estrogen and SHBG in prostate physiology [J]. Prostate, 1996,28(1):17-23.
25. Hudson D L, Guy A T, Fry P, et al. Epithelial cell differentiation pathways in the human prostate. Identification of intermediate phenotypes by keratin expression[J]. J Histochem Cytochem, 2001,49(2):271-278.
26. Risbridger G , Wang H ,Young P,et al. Evidence that epithelial and mesenchymal estrogen receptor-a mediates effects of estrogen on prostatic epithelium[J]. Dev Biol, 2001, 229(2): 432-442.
27. El-Alfy M, Pelletier G, Hermo L S, et al. Unique featuresof the basal cells of human prostate epithelium[J]. Microsc Res Tech,2000,51(5):436-446.
28. Habermann H, Chang W Y, Birch L, et al. Developmental exposure to estrogens alters epithelial cell adhesion and gap junction proteins in the adult rat prostate[J].Endocrinology,2001,142(1):359-369.
29. Kwan P W, Merk F B, Leav I,et al. Estrogen-mediated exocytosis in the glandular epithelium of prostates in castrated and hypophysectomized dogs[J]. Cell Tissue Res ,1982,226(3):689- 693.
30. Aumuller G, Leonhardt M, Janssen M, et al. Neurogenic origin of human prostate endocrinecells[J]. Urology,1999, 53(5):1041-1048.
31. Martin R, Fraile B, Peinado F, et al. Immunohistochemical localization of protein gene product 9.5, ubiquitin, and neuropeptide Yimmunoreactivities in epithelial and neuroendocrine cells from normal and hyperplastic human prostate[J]. J Histochem Cytochem,2000,48(8):1121-1130.
32. Hakanson R, Larsson L I, Sjoberg N O,et al. Amine-producing endocrine-like cells in the epithelium of urethra and prostate of the guinea-pig. A chemical, fluorescence histochemical, and electron microscopic study[J]. Histochemie ,1974, 38(3):259-270.
33. Aumuller G, Renneberg H, Schiemann P J, et al. The role of apocrine releasedproteins in the post-testicular regulation of human sperm function[J]. Adv Exp Med Biol,1997,424:193-219.
34. Kravets F G, Lee J, Singh B, et al. Prostasomes: current concepts[J]. Prostate ,2000, 43(3): 169-174.
35. Saez F, Motta C, Boucher D,et al. Prostasomes inhibit the NADPH oxidase activity of human neutrophils[J]. Mol Hum Reprod,2000, 6(10):883-891.
36. Acosta S, Dizeyi N, Pierzynowski S, et al. Neuroendocrine cells and nerves in the prostate of the guineapig: effects of peripheral denervation and castration[J]. Prostate,2001,46(3): 191-199.
37. Tabassian A R, Nylen E S, Lukacs L, et al. Cholinergic regulation of hamster pulmonary neuroendocrine cell calcitonin[J]. Exp Lung Res, 1990, 16(3):267-277.
38. Simak R, Capodieci P, Cohen D W, et al. Expression of c-kit and kit-ligand in benign and malignant prostatic tissues[J]. Histol Histopathol,2000,15(2): 365-374.
39. Keast J R, Saunders R J. Testosterone has potent, selective effects on the morphology of pelvic autonomic neurons which control the bladder, lower bowel and internal reproductive organs of the male rat[J]. Neuroscience, 1998,85(2): 543-556.
40. Kihara K, Sato K, Ishizaka K, et al. Preservation of ejaculatory function by reconstruction of the canine hypogastric nerve. J Neurosurg 1998,88(4): 726-733.
41. Andersson K E. The concept of uroselectivity. Eur Urol, 1998,33(suppl 2), 7-11.
42. Ruggieri M R, Colton M D, Wang P, et al. Human prostate muscarinic receptor subtypes[J]. J Pharmacol Exp Ther,1995,274(2):976-982.
43. Kostenis E, Zeng FY, Wess J. Structure-function analysis of muscarinic receptors and their associated G proteins[J]. Life Sci,1999,64(6-7):355-362.
44. Onali P, Olianas MC.Bimodal regulation of cyclic AMP by muscarinic receptors. Involvement of multiple G proteins and different forms of adenylyl cyclase[J].life Sci.l995,56(11/12):973-980.
45. Felder cc.Muscarinic acetylcholine receptors:signal transduction through multiple effectors[J].FASEB J,1995,9(8):619-625.
46. Chappie C R, Crowe R, Gilpin S A, et al. The innervation of the human prostate gland—the changesassociated with benign enlargement[J]. J Urol, 1991,146(6): 1637-1644.
47. Hedlund P, Ekstrom P, Larsson B, et al. Heme oxygenase and NO-synthase in the human prostate—relation to adrenergic, cholinergic and peptide-containing nerves[J]. J Auton Nerv Syst,1997,63(3):115-126.
48. Dail W G. Autonomic innervation of male reproductive genitalia.In C. A. Maggi (Ed.), Nervous Control of the Urogenital System[M]. Switzerland: Harwood Academic Publishers, 1993: 69-101
49. Sjostrand N O, Ehren I, Eldh J,et al. NADPH diaphorase in glandular cells and nerves and its relation to acetylcholinesterase- positive nerves in the male reproductive tract of man and guinea-pig[J]. Urol Res, 1998,26(3): 181-188.
50. Dixon J S, Jen P Y,Gosling J A. The distribution of vesicularacetylcholine transporter in the human malegenitourinary organsand its co-localization with neuropeptide Y and nitric oxide synthase[J].Neurourol Urodyn,2000,19(2):185-194.
51. Kaleczyc J, Timmermans J P, Majewski M,et al. Immunohistochemical properties of nerve fibres supplying accessory male genital glands in the pig. A colocalisation study[J]. Histochem Cell Biol,1999,111(3):217-228.
52. Gkonos P J, Guo F, Burnstein K L. Type 1 vasoactive intestinal peptide receptor expression in PC3/AR cells is evidence of prostate epithelial differentiation[J]. Prostate.2000,42(2): 137-144.
53. Reubi J C, Laderach U, Waser B, et al. Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor subtypes in human tumors and their tissues of origin[J]. Cancer Res,2000,60(11): 3105-3112.
54. Lau W A, Pennefather J N, Mitchelson F J. Cholinergic facilitation of neurotransmission to the smooth muscle of the guinea-pig prostate gland[J]. Br J Pharmacol,2000,130(5): 1013-1020.
55. Davis B, Goepel M, Bein S, et al. Lack of neuropeptide Y receptor detection inhuman bladder and prostate[J]. BJU Int,2000,85(7):918-924.
56. Lau W A, Pennefather J N. Muscarinic receptor subtypes in the rat prostate gland[J]. Eur J Pharmacol,1998,343(2-3):151-156.
57. Fernandez J L, Rivera L, Lopez P G, et al. Characterization of the muscarinic receptor mediating contraction of the dog prostate[J]. J Auton Pharmacol, 1998,18(4):205-211.
58. Ellis J, Seidenberg M.. Interactions of alcuronium, TMB-8, and other allosteric ligands with muscarinic acetylcholine receptors: studies with chimeric receptors[J]. Mol Pharmacol,2000,58(6):1451-1460.
59. Pontari M A, Luthin G R, Braverman A S, et al. Characterization of muscarinic cholinergic receptor subtypes in rat prostate[J]. J Recept Signal Transduct Res,1998,18(2-3): 151-166.
60. Hedlund H, Andersson K E, Larsson B. Alpha-adrenoceptors and muscarinic receptors in the isolated human prostate[J]. J Urol,1985,134(6):1291-1298.
61. Caulfield M P. Muscarinic receptors-characterization, couplingand function. Pharmacol Ther,1993,58(3):319-379.
62. Luthin G R, Wang P, Zhou H, et al. Role of ml receptor-G protein coupling in cell proliferation in the prostate. Life Sci ,1997,60(13-14):963-968.
63. Tayebati S K, El-Assouad D, Ricci A,et al. Peripheral blood lymphocytes muscarinic cholinergic receptor subtypes in Alzheimer's disease: a marker of cholinergic dysfunction? [J]. J Neuroimmunol, 2001 ,121(1-2): 126-31
64. Obara K, Arai K, Miyajima N, et al.. Expression of m2 muscarinic acetylcholine receptor mRNA in primary culture of human prostate stromal cells[J]. Urol Res,2000,28(3): 196-200.
65. Mottet N, Costa P, Bali JP. Autonomic nervous system and prostatic physiology. Specific features of the alpha-adrenergic system[J]. Prog Urol,1999,9(1):26-36.
66. Rayford W, Noble M J, Austenfeld M A, et al. Muscarinic cholinergic receptors promote growth of human prostate cancer cells[J]. Prostate ,1997,30 (3):160-166.
67. Wessler I, Kirkpatrick C J, Racke K. The cholinergic 'pitfall': acetylcholine, a universal cell molecule in biological systems, including humans[J]. Clin Exp Pharmacol Physiol, 1999,26(3): 198-205.
68. Eglen R M, Hegde S S, Watson N. Muscarinic receptorsubtypes and smooth muscle function[J]. Pharmacol Rev, 1996,48(4):531-565.
69. Ehlert F J, Ostrom R S, Sawyer G W. Subtypes of muscarinicreceptor in smooth muscle[J]. Life Sci,1997,61(18): 1729-1740.
70. Ehlert F J, Sawyer G W, Esqueda E E. Contractile roles ofM2 and M3 muscarinic receptors in gastrointestinal smooth muscle[J]. LifeSci, 1999, 64(6-7):387-394.
71. Andersson KE. The importance of the cholinergic system in neurourology[J]. Eur Urol, 1998,34( Suppl 1 ):6-9.
72. Todorov L D, Mihaylova-Todorova S T, Bjur R A,. Differential cotransmission in sympathetic nerves: role of frequency of stimulation and prejunctional autoreceptors[J]. J Pharmacol Exp Ther,1999,290(1):241-246.
73. Lau WA, Pennefather JN, Mitchelson FJ. Cholinergic facilitation of neurotransmission to the smooth muscle of the guinea-pig prostate gland[J]. Br J Pharmacol,2000,130(5): 1013-1020
74. Najbar-Kaszkiel A T, Di Iulio J L,et al. Characterisation of excitatory and inhibitory transmitter systems in prostate glands of rats, guinea pigs, rabbits and pigs[J]. Eur J Pharmacol,1997,337(2-3):251-258.
75. Fernandez J L, Rivera L, Lopez P G,et ai. Characterization of the muscarinic receptor mediating contraction of the dog prostate[J]. J Auton Pharmacol, 1998,18(4):205-211
76. Stevens L A, Chappie C R, Chess-Williams R. Human Idiopathic and Neurogenic Overactive Bladders and the Role of M(2) Muscarinic Receptors in Contraction[J]. Eur Urol. 2006 , [Epub ahead of print]
77. Marshall I, Burt R P, Chappie C R. Signal transduction pathways associated with al-adrenoceptor subtypes in cells and tissues including human prostate[J]. Eur Urol, 1999,36 (suppl 1):42-47.
78. McVary K T, Razzaq A, Lee C, et al. Growth of the rat prostate gland is facilitated by the autonomic nervous system[J]. Biol Reprod,1994,51(1): 99-107.
79. Grando S A. Biological functions of keratinocyte cholinergic receptors[J]. J Invest Dermatol Symp Proc ,1997,2(1): 41-48.
80. Klapproth H, Reinheimer T, Metzen J,et al. Non-neuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man[J]. Naunyn Schmiedebergs Arch Pharmacol, 1997,355(4): 515-523.
81. Slater M, Barden J A, Murphy C R. Changes in growth factor expression in the ageing prostate may disrupt epithelial-stromal homeostasis[J]. Histochem J ,2000,32(6):357-364.
82. Prenzel N, Zwick E, Daub H, et al. EGF receptor transactivation by G-proteincoupled receptors requires metalloproteinase cleavage of proHB-EGF[J]. Nature ,1999,402(6764):884-888.
83. Peehl D M, Sellers R G. Induction of smooth muscle cell phenotype in cultured human prostatic stromal cells[J]. Exp Cell Res,1997,232(2):208-215.
84. Yang G, Timme T L, Park S H, et al. Transforming growth factor b1 transduced mouse prostate reconstitutions: I. Induction of neuronal phenotypes[J]. Prostate,1997,33(3):151-156.
85. Dalai R, Djakiew D. Molecular characterization of neurotrophinexpression and the corresponding tropomyosin receptor kinases(trks) in epithelial and stromal cells of the human prostate[J]. Mol Cell Endocrinol ,1997,134(1): 15-22.
86. Pongrac J L, Rylett R J. Molecular mechanisms regulating NGF-mediated enhancement of cholinergic neuronal phenotype: c-fos trans-activation of the choline acetyltransferase gene[J]. J Molec Neurosci, 1998,11(1):79-93.
87. Pflug B R, Dionne C, Kaplan D R,et al. Expression of a trk high affinity nerve growth factor receptor in the human prostate[J]. Endocrinology, 1995, 136(1):262-268.
88. Keast J R, Kepper M E. Differential regulation of trkA and p75 in noradrenergic pelvic autonomic ganglion cells after deafferentation of their cholinergic neighbours[J]. Eur J Neurosci,2001,13(2):211-220.
89. Diaz-Meco M T, Municio M M, Frutos S, The product of par-4, a gene induceduring apoptosis, interacts selectively with the atypical isoforms ofprotein kinase C[J]. Cell,1996,86(5):777-786.
90. Solano R M, Carmena M J, Carrero I, et al. Characterization of vasoactive intestinal peptide/pituitary adenylate cyclase- activating peptide receptors in human benign hyperplastic prostate[J]. Endocrinology, 1996,137(7):2815-2822.
91. Guo Q, Fu W, Xie J, et al. Par-4 is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer's disease[J]. Nat Med, 1998,4(8): 957-962.
92. Guo Q, Xie J, Du H. Par-4 induces cholinergic hypoactivityby suppressing ChAT protein synthesis and inhibiting NGF-inducibility of ChAT activity[J]. Brain Res,2000,874(2):221-232.
93. Lujan M, Paez A, Llanes L, et al. Role of autonomic innervation in rat prostatic structure maintenance: a morphometric analysis[J]. J Urol,1998,160(5): 1919-1923.
94. Doggweiler R, Zermann D H, Ishigooka M,et al. Botox-induced prostatic involution[J]. Prostate ,1998,37(1):44-50.
95. Abbou CC, Salomon L, Chopin D, et al. The current approach to the management of benign hypertrophy of the prostate [J]. Ann Urol,1996,30(6-7):294-301.
96. Keast J R. Effects of testosterone on pelvic autonomic pathways :progress and pitfalls[J]. J Auton Nerv Sys,2000,79(2-3):67-73.
97. Crone J K, Burnett A L, Chamness S L, et al. Neuronal nitric oxide synthase in the canine prostate:aging, sex steroid, and pathology correlations [J]. J Androl,1998,19(3):358-364.
98. Papka R E, Traurig H H, Schemann M,et al. Cholinergic neurons of the pelvic autonomic ganglia and uterus of the female rat: distribution of axons and presence of muscarinic receptors[J]. Cell Tissue Res,1999,296:293-305.
99. Slater M, Barden J A, Murphy C R. Tyrosine kinase A, autonomic and transmitter receptors, but not innervation, are upregulated in the aging rat prostate[J]. Acta Histochem,2000,102(4):427-438.
100. Horsfall D J, Mayne K, Ricciardelli C, et al. Age-related changes in guinea pig prostatic stroma[J]. Lab Invest ,1994,70(5):753-763.
101. Levin R M, Haugaard N, O'Connor L, et al. Obstructive response of human bladder to BPH vs. rabbit bladder response to partial outlet obstruction: a direct comparison[J]. Neurourol Urodyn ,2000,19(5):609- 629.
102. Bayliss M, Wu C, Newgreen D,et al. A quantitative study of atropine-resistant contractile responses in human detrusor smooth muscle, from stable, unstable and obstructed bladders[J]. J Urol,1999,162(5): 1833-1839.
103. Sullivan J, Abrams P. Pharmacological management of incontinence[J]. Eur Urol,1999,36 (suppll): 89-95.
104. Ruffolo R R, Jr, Hieble J P. Adrenoceptor pharmacology: urogenital applications[J]. Eur Urol, 1999,36 (suppl 1):17- 22.
105. Szell E A, Yamamoto T, De Groat W C, et al. Smooth muscle and parasympathetic nerve terminals in the rat urinary bladder have different subtypes of al adrenoceptors[J]. Br J Pharmacol,2000,130(7):1685-1691.
106. Corvin S,Strasser H,Boesch ST, et al. Human rhabdosphincter cell culture:a model for video microscopy of cell contractions[J]. Prostate,2001,47(3): 189-193.
107. Rimmaudo L E,de la Torre E,Sacerdote de Lustig E,et al. Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis[J].Biochem Biophys Res Commun,2005,334(4): 1359-64.
108. Rayford W, Noble M J, Austenfeld M A, et al. Muscarinic cholinergic receptors promote growth of human prostate cancer cells. Prostate[J]. 1997 Feb 15;30(3): 160-166
109. Sydorenko V, Shuba Y, Thebault S. Receptor-coupled, DAG-gated Ca2+-permeable cationic channels in LNCaP human prostate cancer epithelial cells[J]. J Physiol,2003,548(Pt 3):823-836.
110. Blanco M, Robinson M J. Muscarinic M3 acetylcholine receptor immunostaining in paraffin-embedded normal and neoplastic prostatic gland tissue[J]. Ann Diagn Pathol,2004,8(6):333-336.
111. Kester R R, Mooppan U M, Gousse A E,et al. Pharmacological characterization of isolated human prostate[J]. J Urol,2003 ,170(3):1032-1038.
1 Huggins C, Hodges CV. Studies in prostate cancer. I. The effects of castration, of oestrogen, and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res ,1941,1:293-297.
2 Petrylak DP. The current role of chemotherapy in metastatic hormone refractory prostate cancer. Urology, 2005,65 (5 Suppl):3-8.
3 Ganry O. Phytoestrogens and prostate cancer risk. Prev Med,2005,41(1):1-6.
4 Holzbeierlein JM, McIntosh J, Thrasher JB. The role of soy phytoestrogens in prostate cancer. Curr Opin Urol 2005,15(1):17—22.
5 Dalgleish A, Whelan M. Novel immunotherapeutic approaches to prostate cancer. Curr Opin Mol Ther, 2005,7(1):30-34.
6 Tammela T. Endocrine treatment of prostate cancer. J Steroid Biochem Mol Biol, 2004,92(4):287-295.
7 Chang C, Saltzman A, Yeh S, et al.Androgen Receptor: An Overview. Critical Reviews in Eukaryotic Gene Expression, 1995,5(2):97—125.
8 Kyprianou N, English HF, Isaacs JT. Programmed cell death during regression of PC-82 human prostate cancer following androgen ablation. Cancer Res ,1990,50(12):3748-3753.
9 Chen CD, Welsbie DS, Tran C, et al.Molecular determinants of resistance to antiandrogen therapy. Nat Med, 2004,10(1):33-39.
10 Visakorpi T, Hyytinen E, Koivisto P, et al. In vivo amplification of the androgen receptor gene and progression of human prostate cancer. Nat Genet , 1995, 9(4):401-406.
11 Koivisto P, Kononen J, Palmberg C, et al. Androgen receptor gene amplification: A possible molecular mechanism for androgen deprivation therapy failure in prostate cancer. Cancer Res , 1997,57(2):314-319.
12 Eder IE, Culig Z, Ramoner R, et al. Inhibition of LNCaP prostate cancer cells by means of androgen receptor antisense oligonucleotides. Cancer Gene Therapy ,2000,7(7):997-1007.
13 Eder IE, Hoffmann J, Rogatsch H, et al. Inhibition of LNCaP prostate tumor growth in vivo by an antisense oligonucleotide directed against the human androgen receptor. Cancer Gene Therapy, 2002,9(2): 117-125.
14 Ko YJ, Devi GR, London CA, et al.Androgen receptor down-regulation in prostate cancer with phosphorodiamidate morpholino antisense oligomers. J Urol,2004,172(3): 1140-4.
15 Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer. Oncogene, 2003,22(56):9087-9096.
16 Zamore PD, Tuschl T, Sharp PA, et al. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell ,2000,101(1):25-33.
17 Haag P, Bektic J, Bartsch G, et al. Androgen receptor down-regulation by small interference RNA induces cell growth inhibition in androgen sensitive as well as in androgen-independent prostate cancer cells. J Steroid Biochem Mol Biol, 2005,96(3-4):251-258.
18 Liao X, Tang S, Thrasher JB, et al. Small-interfering RNA-induced androgen receptor silencing leads to apoptotic cell death in prostate cancer. Mol Cancer Ther, 2005,4(4):505-515.
19 Soutschek J, Akinc A, Bramlage B, et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature ,2004,432(7014): 173-178.
20 Minamiguchi K, Kawada M, Ohba S, et al.Ectopic expression of the amino-terminal peptide of androgen receptor leads to androgen receptor dysfunction and inhibition of androgen receptor-mediated prostate cancer growth. Mol Cell Endocrinol, 2004,214(1-2): 175-187.
21 Bramlett KS, Dits NF, Sui X, et al. Repression of androgen-regulated gene expression by dominant negative androgen receptors. Mol Cell Endocrinol, 2001,183(1-2): 19-28.
22 Zegarra-Moro OL, Schmidt LJ, Huang H, et al. Disruption of androgen receptor function inhibits proliferation of androgen-refractory prostate cancer cells. Cancer Res, 2002,62(4): 1008-1013.
23 Latham JP, Searle PF, Mautner V, et al. Prostate-specific antigen promoter/enhancer driven gene therapy for prostate cancerxonstruction and testing of a tissue-specific adenovirus vector. Cancer Res, 2000,60(2):334-41.
24 Pang S. Targeting and eradicating cancer cells by a prostate-specific vector carrying the diphtheria toxin A gene. Cancer Gene Ther, 2000,7(7):991-996.
25 Ikegami S, Tadakuma T, Suzuki S, et al. Development of gene therapy using prostate-specific membrane antigen promoter/enhancer with Cre Recombinase/LoxP system for prostate cancer cells under androgen ablation condition. Jpn J Cancer Res ,2002,93(10): 1154-63.
26 Kubo H, Gardner TA,Wada Y, , et al.Phase I dose escalation clinical trial of adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormone-refractory prostate cancer. Hum Gene Ther, 2003,14(3):227-241.
27 Schalken JA, Hessels D, Verhaegh G. New targets for therapy in prostate cancer: differential display code 3 (DD3(PCA3)), a highly prostate cancer-specific gene. Urology ,2003,62(5 Suppl 1):34-43.
28 Farokhzad OC, Jon S, Khademhosseini A, Tran TN, Lavan DA,Langer R. Nanoparticle-aptamer bioconjugates: a new approach for targeting prostate cancer cells. Cancer Res 2004;64:7668-72.
29 Marhefka CA, Gao W, Chung K, et al. Design,synthesis, and biological characterization of metabolically stable selective androgen receptor modulators. J Med Chem, 2004,47(4):993-998.
30 Yin D, Gao W, Kearbey JD, et al. Pharmacodynamics of selective androgen receptor modulators. J Pharmacol Exp Ther, 2003,304(3): 1334-1340.
31 Gao W, Kearbey JD, Nair VA, et al.Comparison of the pharmacological effects of a novel selective androgen receptor modulator, the 5a-reductase inhibitor finasteride,and the antiandrogen hydroxyflutamide in intact rats: New approach for benign hyperplasia. Endocrinology, 2004,145(12):5420-5428.
32 Solit DB, Zheng FF, Drobnjak M, et al. 17-Allylamino-17- demethoxygeldanamycin induces the degradation of androgen receptor and HER-2/neu and inhibits the growth of prostate cancer xenografts. Clin Cancer Res, 2002,8(5):986-993.
33 Vanaja DK, Mitchell SH, Toft DO, et al. Effect of geldanamycin on androgen receptor function and stability. Cell Stress Chaperones ,2002,7(1):55-64.
34 Reed JC. Dysregulation of apoptosis in cancer. J Clin Oncol , 1999, 17(9):2941-53.
35 Raffo AJ, Perlman H, Chen M-W, et al.Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res ,1995,55(19):4438-4445.
36 Yano J, Hirabayashi K, Nakagawa S, et al. Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer. Clin CancerRes, 2004,10(22):7721-7726.
37 Catz SD, Johnson JL. BCL-2 in prostate cancer: a minireview.Apoptosis, 2003,8(1):29-37.
38 Klasa RJ, Gillum AM, Klem RE, et al. Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment. Antisense Nucleic Acid Drug Dev, 2002,12(3): 193-213.
39 Chi KN. Targeting Bcl-2 with oblimersen for patients with hormone refractory prostate cancer. World J Urol ,2005,23(1):33-7.
40 Tolcher AW, Chi K, Kuhn J, et al.A phase II, pharmacokinetic, and biological correlative study of oblimersen sodium and docetaxel in patients with hormone-refractory prostate cancer. Clin Cancer Res ,2005,11(10):3854—61.
41 Morris MJ, Cordon-Cardo C, Kelly WK, et al. Safety and biologic activity of intravenous BCL-2 antisense oligonucleotide (G3139) and taxane chemotherapy in patients with advanced cancer. Appl Immunohistochem Mol Morphol, 2005,13(1):6-13.
42 De Bono JS, Rowinsky EK, Kuhn J, et al. Phase I Pharmacokinetic (PK) and Pharmacodynamic (PD) Trial of bcl-2 antisense (genasense) and docetaxel (D) in hormone refractory prostate cancer. Proceedings of the American Society of Clinical Oncology, 37th Annual Meeting 2001 ;abstract 474.
43 Steinberg J, Oyasu R, Lang S, et al.Intracellular levels of SGP-2 (Clusterin) correlate with tumor grade in prostate cancer. Clin Cancer Res , 1997, 3(10):1707-1711.
44 Pins MR, Fiadjoe JE, Korley F, et al. Clusterin as a possible predictor for biochemical recurrence of prostate cancer following radical prostatectomy with intermediate Gleason scores: a preliminary report. Prostate Cancer Prostatic Dis,2004,7(3):243-248.
45 Gleave M, Miyake H. Use of antisense oligonucleotides targeting the cytoprotective gene, clusterin, to enhance androgen- and chemo-sensitivity in prostate cancer. World J Urol, 2005,23(1):38-46.
46 Springate CM, Jackson JK, Gleave ME, et al. Efficacy of an intratumoral controlled release formulation of clusterin antisense oligonucleotide complexed with chitosan containing paclitaxel or docetaxel in prostate cancer xenograft models. Cancer Chemother Pharmacol, 2005,56(3):239-247.
47 Horiatis D, Wang Q, Pinski J. A new screening system for proliferation-independent anti-cancer agents. Cancer Lett, 2004,210(1):119-24.
48 Tombal B, Denmeade SR, Gillis JM, et al. A supramicromolar elevation of intracellular free calcium ([Ca(2+)](i)) is consistently required to induce the execution phase of apoptosis. Cell Death Differ, 2002,9(5):561-573.
49 Furuya Y, Lundmo P, Short AD, et al. The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin. Cancer Res , 1994, 54(23): 6167-75.
50 Denmeade SR, Jakobsen CM, Janssen S, et al. Prostate-specific antigen-activated thapsigargin prodrug as targeted therapy for prostate cancer. J Natl Cancer Inst, 2003,95(13):990-1000.
51 Denmeade SR, Isaacs JT. The SERCA Pump as a Therapeutic Target:Making a "Smart Bomb" for Prostate Cancer. Cancer Biol Ther, 2005,4(1): 14-22.
52 Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network.Nat Rev Mol Cell Biol, 2001,2(2):127-37.
53 El-Rayes BF, LoRusso PM. Targeting the epidermal growth factor receptor. Br J Cancer 2004;91(3):418-24.
54 Bartlett JM, Brawley D, Grigor K, et al.Type I receptor tyrosine kinases are associated with hormone escape in prostate cancer. J Pathol, 2005,205(4):522-9.
55 Mass RD. The HER receptor family: a rich target for therapeutic development. Int J Radiat Oncol Biol Phys, 2004,58(3):932-940.
56 Lorenzo GD, Bianco R, Tortora G, et al. Involvement of growth factor receptors of the epidermal growth factor receptor family in prostate cancer development and progression to androgen independence. Clin Prostate Cancer, 2003, 2(1):50-57.
57 Zellweger T, Ninck C, Bloch M, et al. Expression patterns of potential therapeutic targets in prostate cancer. Int J Cancer, 2005,113(4):619-628.
58 Signoretti S, Montironi R, Manola J, et al. Her-2-neu expression and progression toward androgen independence in human prostate cancer. J Natl Cancer Inst, 2000,92(23): 1918-1925.
59 Sirotnak FM, She Y, Lee F, rt al. Studies with CWR22 xenografts in nude mice suggest that ZD1839 may have a role in the treatment of both androgen-dependent and androgen-independent human prostate cancer. Clin Cancer Res 2002;8(12):3870-3876.
60 Baselga J, Rischin D, Ranson M, et al. Phase I safety, pharmacokinetic, and pharmacodynamic trial of ZD1839, a selective oral epidermal growth factor receptor tyrosine kinase inhibitor, in patients with five selected solid tumor types. J Clin Oncol, 2002,20(21):4292-302.
61 Lorusso PM. Phase I studies of ZD1839 in patients with common solid tumors. Semin Oncol, 2003,30 (1 Suppl 1):21-29.
62 Canil CM, Moore MJ, Winquist E, et al. Randomized phase II study of two doses of gefitinib in hormone-refractory prostate cancer: a trial of the National Cancer Institute of Canada-Clinical Trials Group. J Clin Oncol . 2005. 23(3):455-4560.
63 Solit DB, She Y, Lobo J, et al. Pulsatile administration of the epidermal growth factor receptor inhibitor gefitinib is significantly more effective than continuous dosing for sensitizing tumors to paclitaxel. Clin Cancer Res, 2005, 11(5):1983—1989.
64 Von Pawel J. Gefitinib (Iressa, ZD1839): a novel targeted approach for the treatment of solid tumors. Bull Cancer, 2004,91 (5):E70-6.
65 Klapper LN, Glathe S, Vaisman N, et al. The ErbB-2/HER2 oncoprotein of human carcinomas may function solely as a shared coreceptor for multiple stroma-derived growth factors. Proc Natl Acad Sci USA, 1999, 96(9): 4995-5000.
66 Hackel PO, Zwick E, Prenzel N, et al. Epidermal growth factor receptors: critical mediators of multiple receptor pathways. Curr Opin Cell Biol, 1999,11(2): 184-9.
67 Klapper LN, Kirschbaum MH, Sela M, et al. Biochemical and clinical implications of the ErbB/HER signaling network of growth factor receptors. Adv Cancer Res, 2000,77:25-79.
68 Baselga J. A new anti-ErbB2 strategy in the treatment of cancer:prevention of ligand-dependent ErbB2 receptor heterodimerization. Cancer Cell, 2002,2(2):93-95.
69 Agus DB, Akita RW, Fox WD, et al. Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell, 2002,2(2): 127-137.
70 Mendoza N, Phillips GL, Silva J, et al. Inhibition of ligand-mediated HER2 activation in androgen-independent prostate cancer. Cancer Res ,2002, 62(19):5485-5488.
71 Craft N, Shostak Y, Carey M, et al. A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat Med 1999;5(3):280-285.
72 Lara Jr PN, Chee KG, Longmate J, et al. Trastuzumab plus docetaxel in HER-2/neu-positive prostate carcinoma: final results from the California Cancer Consortium Screening and Phase II Trial. Cancer ,2004,100(10):2125-2131.
73 Chung LW, Baseman A, Assikis V, et al. Molecular insights into prostate cancer progression: the missing link of tumor microenvironment.J Urol , 2005, 173(1): 10-20.
74 van Moorselaar RJ, Voest EE. Angiogenesis in prostate cancer: its role in disease progression and possible therapeutic approaches. MolCell Endocrinol 2002;197(l-2):239-50.
75 Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell ,1991,64(2):327-36.
76 Folkman J, Shing Y, Angiogenesis. J Biol Chem 1992;267(16): 10931-10934.
77 Jiang WG, Puntis MC, Hallett MB. Molecular and cellular basis of cancer invasion and metastasis: implications for treatment. Br J Surg 1994;81(11): 1576-90.
78 Weidner N, Carroll PR, Flax J,et al. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma.Am J Pathol ,1993,143(2):401-9.
79 Jackson MW, Roberts JS, Heckford SE, et al. A potential autocrine role for vascular endothelial growth factor in prostate cancer. Cancer Res, 2002,62(3):854-9.
80 Doll JA, Reiher FK, Crawford SE, et al. Thrombospondin-1, vascular endothelial growth factor and fibroblast growth factor-2 are key functional regulators of angiogenesis in the prostate. Prostate 2001;49(4):293-305.
81 Chan LW, Moses MA, Goley E, et al. Urinary VEGF and MMP levels as predictive markers of 1 -year progression-free survival in cancer patients treated with radiation therapy: a longitudinal study of protein kinetics throughout tumor progression and therapy. J Clin Oncol, 2004,22(3):499-506.
82 Melnyk O, Zimmerman M, Kim KJ, et al. Neutralizing antivascular endothelial growth factor antibody inhibits further growth of established prostate cancer and metastases in a pre-clinical model. J Urol ,1999,161(3):960-3.
83 Petrylak DP. Future directions in the treatment of androgen-independent prostate cancer. Urology ,2005,65 (6 Suppl):8-12.
84 Figg WD, Kruger EA, Price DK, et al. Inhibition of angiogenesis: treatment options for patients with metastatic prostate cancer. Invest New Drugs ,2002,20(2): 183-94.
85 Nicholson B, Guiding K, Conaway M, et al.Combination antiangiogenic and androgen deprivation therapy for prostate cancer: a promising therapeutic approach. Clin Cancer Res, 2004,10(24):8728-8734.
86 Pettit GR, Rhodes MR, Herald DL, et al. Antineoplastic agents. 445. Synthesis and evaluation of structural modifications of (Z)- and (E)-combretastatin A-41. J Med Chem, 2005,48(12):4087-99.
87 Leblanc R, Dickson J, Brown T, et al. Synthesis and cytotoxicity of epoxide and pyrazole analogs of the combretastatins. Bioorg Med Chem, 2005, 13(21):6025-6034
88 Siemann DW, Bibby MC, Dark GG, et al. Differentiation and definition of vascular-targeted therapies.Clin Cancer Res ,2005,11(2 Pt 1):416—20.
89 Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature ,1988,322(6163):411-5.
90 Battistini B, Chailler P, D'Orleans-Juste P, et al. Growth regulatory properties of endothelins. Peptides ,1993.14(2):385-399.
91 Vacca F, Bagnato A, Catt KJ, et al. Transactivation of the epidermal growth factor receptor in endothelin-1-induced mitogenic signaling in human ovarian carcinoma cells. Cancer Res ,2000,60(18):5310-7.
92 Nelson JB. Endothelin receptor antagonists. World J Urol ,2005,23(1):19-27.
93 Jimeno A, Carducci M. Atrasentan: a novel and rationally designed therapeutic alternative in the management of cancer. Expert Rev Anticancer Ther, 2005,5(3):419-27.
94 Stewart DA, Cooper CR, Sikes RA. Changes in extracellular matrix (ECM) and ECM-associated proteins in the metastatic progression of prostate cancer. Reprod Biol Endocrinol 2004;2:2.
95 Kleiner DE, Stetler-Stevenson WG. Matrix metalloproteinases and metastasis. Cancer Chemother Pharmacol 1999;43 Suppl:S42-51.
96 Trudel D, Fradet Y, Meyer F,et al. Significance of MMP-2 expression in prostate cancer: an immunohistochemical study. Cancer Res, 2003,63(23): 8511-5.
97 Zhang L, Shi J, Feng J, et al. Type IV collagenase (matrix metalloproteinase-2 and -9) in prostate cancer.Prostate Cancer Prostatic Dis, 2004,7(4):327-32.
98 Knox JD, Bretton L, Lynch T, et al. Synthetic matrix metalloproteinase inhibitor, BB-94, inhibits the invasion of neoplastic human prostate cells in a mouse model. Prostate, 1998,35(4):248-54.
99 Somlyo AV, Phelps C, Dipierro C, et al. Rho kinase and matrix metalloproteinase inhibitors cooperate to inhibit angiogenesis and growth of human prostate cancer xenotransplants.Faseb J 2003 ;17(2):223-34.
100 Rosenbaum E, Zahurak M, Sinibaldi V, et al. Marimastat in the treatment of patients with biochemically relapsed prostate cancer: A prospective randomized, double-blind,phase I/II trial. Clin Cancer Res ,2005,11(12):4437-43.
101 Coussens LM, Fingleton B, Matrisian LM. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science, 2002,295(5564):2387-92.
102 Shamsuddin AM, Vucenik I, Cole KE. IP6: a novel anti-cancer agent.Life Sci, 1997,61(4):343-54.
103 Agarwal C, Dhanalakshmi S, Singh RP, et al. Inositol hexaphosphate inhibits growth and induces Gl arrest and apoptotic death of androgen-dependent human prostate carcinoma LNCaP cells.Neoplasia 2004;6(5):646-59.
104 Singh RP, Sharma G, Mallikarjuna GU, et al. In vivo suppression of hormone-refractory prostate cancer growth by inositol hexaphosphate: induction of insulin-like growth factor binding protein-3 and inhibition of vascular endothelial growth factor. Clin Cancer Res, 2004,10(1 Pt 1)::244-250.
105 Adams J, Palombella VJ, Sausville EA, et al. Proteasome inhibitors: A novel class of potent and effective antitumor agents. Cancer Res, 1999,59(11): 2615-2622.
106 Herrmann JL, Briones Jr F, Brisbay S, et al.Prostate carcinoma cell death resulting from inhibition of proteasome activity is independent of functional bcl-2 and p53. Oncogene ,1998,17(22):2889-99.
107 Williams SA, McConkey DJ. The proteasome inhibitor bortezomib stabilizes a novel active form of p53 in human LNCaP-Pro5 prostate cancer cells. Cancer Res, 2003,63(21):7338-44.
108 Williams SA, Pettaway C, Song R, et al. Differential effects of the proteasome inhibitor bortezomib on apoptosis and angiogenesis in human prostate tumor xenografts. Mol Cancer Ther, 2003,2(9):835-43.
109 Papandreou CN, Daliani DD, Nix D, et al. Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer. J Clin Oncol 2004;22(11):2108—21.
110 Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell, 1994,79(1): 13-21.
111 Price N, Dreicer R. Phase I/II trial of bortezomib plus docetaxel in patients with advanced androgen-independent prostate cancer. Clin Prostate Cancer 2004;3(3): 141-3.
112 Hidalgo M, Rowinsky EK. The rapamycin-sensitive signal transduction pathway as a target for cancer therapy. Oncogene 2000;19(56):6680-6686.
113 Wu L, Birle DC, Tannock IF. Effects of the mammalian target of rapamycin inhibitor CCI-779 used alone or with chemotherapy on human prostate cancer cells and xenografts. Cancer Res, 2005,65(7):2825-2831.
114 Iversen PL, Arora V, Acker AJ, et al. Efficacy of antisense morpholino oligomer targeted to c-myc in prostate cancer xenograft murine model and a Phase I safety study in humans. Clin Cancer Res, 2003,9(7):2510-9.
115 Devi GR, Beer TM, Corless CL, et al. In vivo bioavailability and pharmacokinetics of a c-MYC antisense phosphorodiamidate morpholino oligomer, AVI-4126, in solid tumors. Clin Cancer Res ,2005,11(10):3930-3938.
116 Lee Y, Vassilakos A, Feng N, et al.GTI-2040, an antisense agent targeting the small subunit component(R2) of human ribonucleotide reductase, shows potent antitumor activity against a variety of tumors. Cancer Res, 2003,63(11): 2802-11.
1 1 Pennefather J N, Lau WA, Mitchelson F,et al. The autonomic and sensory innervation of the smooth muscle of the prostate gland: a review of pharmacological and histological studies. J AutonPharmaco,2000, 20(4): 193-206.
2 Ruggieri MR, Colton MD, Wang P, et al.Human prostate muscarinic receptor subtypes. J Pharmacol Exp Ther ,1995,274 (2) : 976-982.
3 Batra S, Christensson PI,Hartley-Asp B. Characterization of muscarinic cholinergic receptors in membrane preparations from rat prostatic adenocarcinoma. Prostate, 1990,17(4): 261-268.
4 of smooth muscle cells from human prostate. J Urol, 1994,152 (6) : 2173- 2177.
5 Obara K, Arai K, Miyajima Ret al..Expression of m2 muscarinic acetylcholine receptor mRNA in primary culture of human prostate stromal cells. Urol Res, 2000,28(3): 196-200.
6 Fernandez JL,Rivera L,Lopez PG,Recio P,et al. Characterization of the muscarinic receptor mediating contraction of the dog prostate. J Auton Pharmacol, 1998,18(4):205-11.
7 Cheng K, Chen Y , Zimniak P,et al. Functional interaction of lithocholic acid conjugates with M3 muscarinic receptors on a human colon cancer cell line. Biochimica et Biophysica Acta , 2002,1588 (1) : 48-55.
8 Rimmaudo LE,de la Torre E,Sacerdote de Lustiq E,et ai.Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis. Biochem Biophys Res Commun,2005,334(4): 1359-64.
9 Luthin G R, Wang P, Zhou H, et al. Role of ml receptor-G protein coupling in cell proliferation in the prostate. Life Sci, 1997,60(13-14): 963- 968.
10 Fiszman G , Cattaneo V , Torre, E, et al. Muscarinic receptors autoantibodies purified from mammary adenocarcinoma-bearing mice sera stimulate tumor progression. International Immunopharmacology, 2006,6( 8): 1323-1330.
11 Umeda E, Aramaki Y. Mori T.et al. Muscarinic receptor subtypes modulate the release of [3H]-noradrenaline in rat spinal cord slices. Brain Research Bulletin. 2006,70 (1) :99-102.
12 Blanco M,Robinson MJ.Muscarinic M3 Acetylcholine Receptor Immunostaining in Paraffin-Embedded Normal and Neoplastic Prostatic Gland Tissue. Annals of Diagnostic Pathologly, 2004,8(6):333-336.
1 Gasparini G,Bevilacqua P ,Bonoldi E , et al . Predictive and prognosis markers in a series of patient s with head and neck squamous cell invasive carcinoma treated with concurrent chemoradiation therapy [J ] . Clin Can Res , 1995 , 1(1): 1375-1383.
2 Hanahan D ,Folkman J . Patterns and emerging mechanism of the angiogenic switch during tumorigenesis [J ] . Cell ,1996 ,86 (3) : 353-364.
3 Kaushal V ,Mukunyadzi P ,Dennis RA , et al . Stage2specific characterization of the vascular endothelial growth factor axis in prostate cancer : expression of lymphangiogenic markers is associated with advanced2stage disease[J ] . Clin Cancer Res ,2005 ,11 (2Pt 1) :584-593.
4 Latil A ,Bieche I , Pesche S , et al . VEGF overexpression in clinically localized prostate tumors and neuropilin21 overexpression in metastatic forms[J ] . Int J Cancer ,2000 ,89 (2) : 1672171.
5 Feldman A L, Tamarkin L, Paciotti G F, et al.Serum endostatin levels are elevated and correlate with serum vascular endothelial growth factor levels in patients with stage IV clear cell renal cancer [J]. Clin Cancer Res, 2000, 6 (12) : 4628- 4634.
6 Kugler A,Hemmerlein B, Thelen P, et al.Expression of metalloproteinase 2 and 9 and their inhibitors in renal cell carcinoma[J]. J Urol, 1998, 160 (5) .1914- 1918.
7 Sasaki T , Fukai N ,Mann K, et al. Structure, function and tissue forms of the C2terminal globular domain of collagen containing the angiogenesis inhibitor endostatin [ J ] . EMBO J , 1998 , 17(15): 4249-4256.
8 Cheng K, Chen Y , Zimniak P,et al. Functional interaction of lithocholic acid conjugates with M3 muscarinic receptors on a human colon cancer cell line. Biochimica et Biophysica Acta, 2002,1588 (1) : 48-55.
9 Fiszman G , Cattaneo V , Torre, E, et al. Muscarinic receptors autoantibodies purified from mammary adenocarcinoma-bearing mice sera stimulate tumor progression. International Immunopharmacology, 2006,6( 8): 1323-1330.
10 Umeda E, Aramaki Y, Mori T,et al. Muscarinic receptor subtypes modulate the releaseof [3H]-noradrenaline in rat spinal cord slices. Brain Research Bulletin, 2006: 70 (1) ,99-102.