食管神经和递质对食管癌细胞增殖与分化的作用及其机制
|
摘要
1研究背景
河南林州(原林县)及其毗邻的地区是世界上食管癌(esophagaeal cancer，EC)发病率和死亡率最高的地区，而且，EC当前仍是该地区肿瘤相关疾病的主要死亡原因。但是，目前食管癌变的分子机制仍不十分清楚。最近有文献报道食管癌的发生发展与个体的心理应激、神经-免疫因素等密切相关。
神经系统可经中枢—周围自主神经(交感神经和迷走神经等)通路调节内脏效应细胞。有研究报道，食管闭锁患者迷走神经和喉返神经发育不良；动物实验证实，去除颈段神经嵴或颈交感神经节均可导致食管黏膜缺如和食管闭锁，表明食管与其神经之间在结构和功能上存在着密切关系。近年的临床研究发现，食管癌切除术保留迷走神经，可使患者术后恢复加快，肿瘤复发延迟。西方学者进行大量的研究认为，食管运动功能紊乱所致胃．食管反流可能是导致食管腺癌发生的危险因素之一。这些研究提示，迷走神经功能紊乱或缺陷可能在食管癌变中起着重要的作用。
迷走神经的副交感纤维和交感神经通过释放乙酰胆碱(Acetylcholine，ACh)和去甲肾上腺素(Norepinephrine，NE)及其受体(Acetylcholine receptor，AChRs)实现对食管的功能调节。神经生物学研究发现AChRs对细胞增殖、分化、细胞骨架形成具有重要的生理作用，其激动剂尼古丁可促进胶质细胞肿瘤的细胞增殖，而拮抗剂筒箭毒则具有抑制细胞增殖的效应。已有临床调查提示，恶性肿瘤发生与mAChR功能异常有关。还有报道NE可调节中枢神经元的分化类型，亦可诱导人的肺泡细胞系凋亡。但有关神经及其递质对食管癌变的影响，尚未见报道。我们认为食管癌变及其演进是通过多因素、多阶段、多基因的作用，且具有复杂的机制。本实验室多年的研究也提示多个抑癌基因和癌基因以及蛋白质的功能紊乱是导致食管癌变始动的原因，但尚未能发现关键的基因与蛋白分子。食管癌变的好发部位为食管中下段，食管癌前病变并具有双向发展的不稳定特性，提示食管癌变具有可逆性。另有研究发现激活脑干5-HT中缝核—迷走神经通路对内脏具有保护作用，表明中缝核5-HT神经元通过释放5-HT调节着迷走神经的功能。有关心理应激(Stress)的研究发现，长期的心理压力或抑郁可导致脑内5-HT及其受体水平降低，提示应激可影响迷走神经至食管的胆碱能功能下降，进而推测食管病理变化可能与迷走神经至食管神经通路的结构和功能异常有关，而且食管神经功能异常在食管癌变与食管癌细胞恶性转化中占有重要地位。近年的研究提示，EC细胞角质蛋白(Keratin)、氧化还原酶(PRX1)、钙依赖磷脂结合蛋白(Annexin-2)表达异常。Keratin 5和Keratin 20分别为上皮祖细胞和成熟细胞的标志，也是上皮细胞起黏附、支持和保护的功能基础。PRX1对细胞DNA损伤具有修复作用。Annexin-2具有调节细胞分化和抑制细胞增殖的作用等，可能是抑制食管癌变的重要分子之一。AChR和胆碱乙酰基转移酶(CHAT)是ACh作用于细胞的传递分子。微管相关蛋白(MAP2)是神经元的骨架连接蛋白，退行性变的神经元及神经纤维表达减少。上述分子为进一步研究提供了重要的线索和观察的指标。
因此，本研究采用组织切片、HE染色、免疫荧光、免疫组化染色等方法对食管癌壁间神经丛的形态与食管癌细胞功能基因与蛋白表达变化进行观察，对其与食管癌变和癌细胞转化的关系予以分析。并进一步采用体外细胞培养法、免疫荧光与免疫组化染色、流式细胞以及RT-PCR技术等探讨交感和副交感神经及其递质NE与ACh对食管癌细胞抑癌基因和功能蛋白表达变化的影响，分析ACh与NE对食管癌变与癌细胞转化的作用，旨在揭示与阐明食管神经及其递质变化在食管癌变中的作用和意义，并为食管癌的靶向防治提供新的研究思路。
2材料与方法
2．1 EC壁间神经形态与癌细胞功能蛋白表达变化关系的观察
42例食管癌标本均来自河南省林州市中心医院和姚村食管癌医院，男性37例，女性5例，其平均年龄为60.3±6.5。所有患者在术前均未接受任何化疗或放疗。经4％的多聚甲醛固定、石蜡包埋、连续切片，采用免疫荧光、免疫组化ABC法，对癌、癌旁和正常组织进行免疫组化染色，观察与分析癌、癌旁和正常组织壁间神经形态与癌细胞AChR、ChAT、Annexin-2、PRX1、MAP2、Keratin5和Keratin20表达变化之间以及各功能蛋白表达变化相互之间的关系，以了解食管神经在食管癌变过程中的作用及其变化特点。
2．2体外细胞培养实验
采用细胞培养法，并借助细胞受体阻断剂(阿托品、托拉唑林)、细胞外基质阻断剂(U0126)对人食管癌细胞株EC9706细胞(郑州大学医学院癌症研究室所保存食管鳞癌细胞株)进行体外培养，应用免疫组化、免疫荧光、MTT、平板细胞克隆、流式细胞、荧光定量RT-PCR等技术方法，观察ACh、NE处理前后EC9706细胞的AChR、ChAT、Annexin-2、Keratin 5和Keratin 20等表达以及细胞增殖周期与增殖活力的变化，分析ACh、NE以及细胞外基质对食管癌细胞增殖和分化的影响与相互关系，以揭示神经递质对食管癌变、癌细胞转化的作用及其可能性分子机制。
2．3统计学分析
实验数据采用SPSS10.0统计学软件处理，选用单因素方差分析和配对样本t检验。数据资料用(?)±s表示，以a-0.05为检验水准，P＜0.05表示差异有显著意义，P＜0.01表示差异有非常显著意义。
3结果
3．1食管壁间神经形态与功能蛋白表达的变化及其关系
食管壁间神经MAP2阳性强度由正常到增生、原位癌、癌逐渐减淡，细纤维减少至丧失。AChR、ChAT、Annexin-2、PRX1、K20表达随癌变演进和食管壁间神经退行变的加重逐步减少，Keratin 5在癌变过程中变化较小。Keratin 20、Annexin-2的表达变化与PRX1表达一致。AChR表达变化的特征是随癌变的演进开始由胞膜、胞浆随即逐步减少，而后从胞质向核内集中或转移。ChAT在癌变及其恶性转化过程中变化与减少较晚，非典型增生阶段呈现过表达现象。
3.2 ACh与NE对人食管癌细胞系细胞增殖、分化的影响
3．2．1 ACh和NE对体外培养食管癌细胞系EC9706细胞生长形态的影响
应用ACh和NE培养后，可促使细胞体增大，突起长出，核质比下降；NE可使体外培养的食管癌细胞密度降低，细胞突起更长。ACh和NE两组的突起长度与其余各组相比均具有非常显著性差异(P＜0.01)。在mAChR和a受体阻断后，ACh和NE仍具有促进细胞突起生长的作用，两组与对照组相比具有显著性差异(P＜0.05)。ACh和NE组核质比与对照组相比具有显著性差异(P＜0.05)；ACh、NE组受体阻断前、后相比，核质比有所增大，但无显著性差异(P＞0.05)。
3．2．2 ACh和NE对体外培养食管癌细胞系EC9706细胞增殖活性的影响
ACh和NE均具有降低体外培养食管癌细胞增殖活性的作用，与对照组相比具有显著性差异(P＜0.05)；mAChR和a受体阻断后，仍保留有部分降低体外培养食管癌细胞增殖活力的作用。
3．2．3 ACh和NE对体外培养食管癌细胞系EC9706细胞增殖抑制率的的影响
ACh、NE可提高体外培养食管癌细胞系EC9706细胞增殖抑制率，对细胞增殖具有抑制作用，与对照组相比具有差异显著性(P＜0.05)。受体阻断后，集落形成抑制率仍下降。
3．2．4 ACh和NE对体外培养食管癌细胞系EC9706细胞AChR、ChAT、PRX1、Annexin-2、Keratin5和K20表达的影响
ACh和NE可促进体外培养食管癌细胞系EC9706细胞的AChR、ChAT、PRX1、Annexin-2、Keratin5和K20表达明显增多，与对照组相比具有显著性差异(P＜0.05)；在mAChR和a受体阻断后，仍保留有部分促进体外培养食管癌细胞功能蛋白表达的作用，其中ACh经阿托品阻断mAChR后，与对照组相比，仍较对照组为高，具有统计学意义(P＜0.05)。
3．2．5 ACh和NE对体外培养食管癌细胞系EC9706细胞周期与凋亡的影响
ACh和NE对细胞周期具有阻滞作用，可使细胞阻滞于G1期或G2期，与对照组相比可使S期癌细胞数量减少，阻滞作用由强到弱，依次为：ACh+NE＞NE加托拉唑林＞NE＞ACh加阿托品＞ACh，阻滞率分别占对照组的90％、70％、15％、15％、10％。ACh和NE具有诱导体外培养食管癌细胞凋亡的作用，诱导癌细胞凋亡率分别是对照组的30倍(ACh)、4倍(ACh加阿托品)、10倍(NE)、20倍(NE+托拉唑啉)、30倍(ACh+NE)。
3．2．6 ACh和NE对食管癌细胞系EC9706细胞Annexin-2 mRNA表达的影响
ACh和NE可促使体外培养食管癌细胞系EC9706细胞Annexin-2 mRNA表达增多，与对照组相比具有显著性差异(P＜0.05)；ACh、ACh+阿托品、ACh+NE、NE、NE+T的Annexin-2 mRNA表达水平分别比对照组增高20倍、8倍、3倍、2倍、和4倍。
3．3 ACh和NE对阻断ERK1／2食管癌细胞系EC9706细胞增殖和分化的影响
阻断ERK1／2体外培养食管癌细胞系EC9706细胞形态的突起变短，核质比增大，与对照组相比均有显著差异(P＜0.05)。ACh和NE可使ERK1／2阻断后细胞核质比下降，具有统计学意义(P＜0.05)；变短的突起稍长，但无显著差异(P＞0.05)。阻断ERK1／2体外培养食管癌细胞系EC9706细胞的AChR、ChAT、Annexin-2、Keratin20表达均显著减少(P＜0.05)，加用ACh和NE可使下降的AChR、ChAT、Annexin-2、Keratin20表达增多，具有统计学意义(P＜0.05)。阻断ERK1／2食管癌细胞系EC9706可使细胞的增殖活性下降，细胞增长抑制率提高(P＜0.05)，加用ACh和NE使ERK1／2降低的细胞增殖活性再度下降，细胞增长抑制率再度提高。阻断ERK1／2使体外培养食管癌细胞系EC9706细胞数目在S期阻滞与增多，凋亡细胞百分数减少，ACh和NE可使由ERK1／2阻断引起的S期细胞百分数降低，其作用由强到弱分别为ACh+NE＞ACh+A＞ACh＞NE+托拉唑林＞NE。
4结论
4．1食管壁间神经纤维在癌变演进过程中逐渐减少或丧失，AChR、ChAT、Annexin-2、PRX1、K20随癌变的表达逐步减少。结果提示，食管壁间神经减少或功能丧失可能是食管癌变的神经因素。AChR随癌变始动与演进的特征性变化是其在胞膜、胞浆的含量减少最早，并从胞质向核内集中或转移，这可能是引起食管黏膜上皮细胞对神经性ACh敏感度下降，进而导致癌变启动较早的受体事件。PRX1表达增多可能是受伤害刺激的细胞维持Annexin-2和Keratin20表达有效分子，其表达下降可能是导致细胞自身保护和稳定功能下降，细胞癌变与癌细胞恶化的内在原因。
4．2ACh和NE具有诱导体外培养的人食管癌细胞系EC9706细胞分化的作用，采用阿托品和妥拉唑啉分别阻断mAChR和α受体，仍具有促进EC9706细胞分化的作用。结果提示，食管癌细胞具有经诱导分化或增强分化的能力，ACh和NE可促使细胞体增大，突起长出，核质比下降，功能蛋白表达增多，其机制可能是主要通过nAChR和β受体。
4．3 ACh和NE对体外培养食管癌细胞系EC9706细胞增殖活性具有降低的作用，表现为将癌细胞由S期向G1和G2期诱导，或使其周期发生阻滞，影响细胞的增殖，该作用经多受体、多途径韵调节。
4．4 ACh和NE对体外培养食管癌细胞系EC9706细胞Annexin-2 mRNA表达具有促进作用。ACh作用最强，可使其表达量增加20倍，ACh+阿托品使之增加8倍，NE、NE+托拉唑啉、ACh+NE作用较弱，仅能使之表达增加2～4倍。
4．5 ERK1／2阻滞剂除具有降低细胞增殖和阻滞细胞分化的作用。ACh和NE对阻断ERK1／2体外培养食管癌细胞系EC9706细胞的增殖活性具有再度降低的作用，并通过调节癌细胞功能蛋白的表达而诱导细胞分化。神经递质与细胞外基质阻断剂相互作用的发现为进一步揭示食管癌在体内发生发展的分子机制提供了新的研究思路和方向，并为食管癌防治的药物设计提供了新的靶点。
1 BACKGROUND
Linzhou City (formerly Linxian County), together with nearby counties in Henan,has been well recognized as the highest morbidity and mortality area for esophagealcancer (EC) in the world. Moreover, EC remains currently the leading cause ofcancer-related death in these areas. Nevertheless, the molecular mechanism ofesophageal carcinogenesis is largely unknown. Recent studies have indicated that thegeneration and development of EC are correlated with the individual's psychologicalstress and neuroimmunologic factors.
The viscera effector cells can be regulated by the nervous system viacenter-peripheral autonomic nervous (sympathetic nerve and vagus nerve) pathway. Ithas been reported that there is hypogenesis of vagus nerve and recurrent laryngealnerve in esophageal atresia patients. And animal experiments also demonstrated thatthe removal of either cervical nervous crest or cervical sympathetic ganglia can leadto esophageal mucosa absence and esophageal atresia, which indicates thatesophageal diseases are closely related to esophageal nerves both in structure and function. Recent clinical researches indicate that EC exsection with vagus nervereserved could accelerate postoperative recovery and procrastinate the tumor relapse.Western scholars have carded out a great deal of researches and presume thatgastroesophageal reflux disease (GERD) resulting from functional disorder ofesophageal motor may contribute to esophageal carcinogenesis, which indicates thatthe functional disorder or defection of vagus nerve may play an important role inesophageal carcinogenesis.
The acetylcholine (ACh) and norepinephrine (NE) released fromparasympathetic fibers of vagus nerve and sympathetic fibers and their receptors carryout the regulation of esophageal function. Neurobiological researches demonstrate thatacetylcholine receptors (AChRs) have significant physiological functions on cell proliferation,differentiation and formation of cytoskeleton. Nicotine, the excitomotor of AChRs, prompts theproliferation of gliacyte tumor while tubocurarine (TC) the antagon inhibits that. Clinicalinvestigations have shown that the generations of malignant tumors were correlatedwith dysfunction of mAChR. And it is also reported that NE can regulate the differential typeof central neuron and induce the apoptosis of alveolar cell. But there is no report about the effectof nerves and corresponding transmitters on esophageal carcinogenesis. We presume thatesophageal carcinogenesis is a multistage progressive process involved by multiple factors,multiple genetic changes and their complicated mechanism. Researches of years in ourlaboratory also indicate that a great deal of anti-oncogenes and oncogenes and the functionaldisorders of proteins are the original causes of esophageal carcinoma, but the key genes andprotein molecules haven't been identified. It is discovered from recent census andfollow-up visits that the predilection sites for esophageal carcinogenesis are the middleand inferior segments, and the character of precancerous lesions are of two-waydevelopment, which indicates that the carcinogenesis is reversible. Other studies which find thatthe activation of the brainstem 5-HT (5-hydroxytryptamine, 5-HT) rapheal nuclei-vagus pathwaycan protect the viscera manifested that the rapheal nuclei 5-HT neuron regulates the vagus nerveby releasing 5-HT. Researches on psychological stress discovered that long-standing mental stressor depression leads to descending of center 5-HT and its receptor level, indicating that stress maydegrade the cholinergic function from vagus nerve to esophagus, thus it can be inferred that the esophageal pathological changes may concern the structural and functional abnormality of theneural pathway from vagus nerve to esophagus, and that the disfunction of the esophageal nervetakes a significant place in esophageal cancerization and the malignant transformation ofesophageal carcinoma cells. Recent studies have indicated that EC cell has the feature ofabnormally expressing Keratins, Peroxiredoxin-1 (PRX1) and calcium-dependent lipid bindingprotein-2 (Annexin-2). Keratin 5 (KS) and Keratin 20 (K20) are the marks of progenitorepithelium cells and mature cells respectively, and they are also the foundation of epithelium celladhering, sustaining and protecting functions. PRX1 has repairing function to damaged DNA;Annexin-2 can suppress cells' proliferation and regulate cells' differentiation and it may be one ofthe important molecules inhibiting esophageal carcinogenesis. AChR and Cholineacetyltransferase (CHAT) are transmitting molecules by which ACh acts on cells.Microtubule-associated protein-2 (MAP2) is a kind of skeleton connection protein in the neuron,the expression of which may be decreased in degenerating neurons and nerve fibers.These molecules mentioned here are provided as observational indexes for furtherresearch.
Therefore, the design of this research adopts the methods and techniques of tissues sliced, HEstraining, immunofluorescence (IMF), immunohistochemical straining and so on to survey thedistribution features of esophageal nerves, the intramural neuroplexus morphous of the esophagealcarcinoma, and the changes of the functional genes and protein expression, and to analyze therelationship between those indexes mentioned above and the carcinogenesis and celltransformation.Furthermore, we use vitro cell culture, immunofluorescence, immunohistochemicalstraining, flow cytometry, RT-PCR etc. to make an exploratory study on the affection of NE, thesympathetic transmitter, and ACh, the parasympathetic transmitter, on the changes inanti-oncogenes and functional protein expression of the esophageal carcinoma, thus we cananalyze the effects of NE and ACh on esophageal carcinogenesis and cell transformation, which isof significance in revealing and illuminating the molecular mechanism of esophagealcarcinogenesis, and providing new research ideas on target for cancer prevention and treatment.
2 MATERIAL and METHODS
2.1 Observation of the relationship between EC intramural nerve morphous and expression changes of cancer cellular functional proteins.
There were 42 EC specimens that were all from Central Hospital in Linzhou orYaocun EC Hospital and none of the patients had accepted any chemotherapy orradiotherapy. The 42 patients included 37 males and 5 females, whose mean age was60.3±6.5. After 4% polyformalin fixation, paraffin imbedding, serial sectioning anddying the cancer tissue, para (adjacent) carcinoma tissue and normal tissue via IMFand immunohistochemical ABC method, we surveyed and analyzed the relationsamong intramural nerve morphous of the three kinds of tissues and AChR, CHAT,Annexin-2, PRX1, MAP2, Keratin 5 and Keratin 20 expression changes of the cancercells, including relations among expression changes of every functional proteins, so asto identify the change pattern and the effect of esophageal nerves in the process ofesophageal carcinogenesis.
2.2 Experiment of cell culture in vitro
By adding cell receptor blocking agents Atropine and Tolazoline and xtracellularmatrix (ECM) blocking agent U0126, we cultured in vitro the human esophagealcarcinoma cell strain EC9706, the ESCC (esophageal squamous carcinoma cell,ESCC) strain preserved in the Cancer Lab of the Medical College of ZhengzhouUniversity. By means of immunohistochemical straining, IMF, flat cell clone, flowcytometry, fluorescently quantitative RT-PCR and so on, we observed the changes ofthe cell cycle and vigor of cell generation as well as the changes of the expression ofAChR, CHAT, Annexin-2, Keratin 5 and Keratin 20 in EC9706 both before and afterthe treatment of ACh and NE, then analyzed the affection of ACh, NE and ECM to theproliferation and differentiation of EC cells and their mutual relationship, so as todetect the effect of neurotransmittcr in the process of esophageal carcinogenesis andcancer cell transformation and find the possible molecular mechanism.
2.3 Statistics analysis
The data is analyzed with SPSS10.0 by means of one-factor analysis of varianceand paired sample t-test. It is described as (?)±s, with a=0.05 as the size of test andP＜0.05 meaning the variance to be significant.
3 RESULTS
3.1 The relation between esophageal intramural nerve morphous and expressionchanges of cancer cellular functional proteins
The MAP2 positive intensity of esophageal intramural nerve decreases graduallyin the order of normal tissue, hyperplasia, preinvasive carcinoma and cancer, whilethe fine nerve fibers reduce and finally disappear. The expressions of AChR, CHAT,Annexin-2, PRX1, and Keratin 20 decrease gradually along with the development ofcarcinogenesis and the aggravation of esophageal intramural nerve retrogradation,while Keratin5 changes less in the process of carcinogenesis. The change ofKeratin20、Annexin-2 expression keeps the same with PRX1. The expression ofAChR is typically to decrease gradually from cell membrane to cytoplasm and thenshift or concentrate from cytoplasm into nuclear with the development ofcarcinogenesis. ChAT changes and decreases late in the process of carcinogenesis" andmalignant transformation and expresses excessively in the atypical hyperplasia phase.
3.2 The effect of ACh and NE on the cell proliferation and differentiation ofEC9706, the human esophageal carcinoma cell line cultured in vitro
3.2.1 The effect of ACh and NE on the cell proliferation of EC9706, theesophageal carcinoma cell line in vitro
After treatment of ACh and NE, the cell soma was augmented, the processeserupted and the nuclear-cytoplasmic ratio was decrease. The esophageal carcinomacells grow with lower density and have longer processes under the role of NE in vitro.The process length of ACh and NE groups have highly significant differencecompared with the other groups (P＜0.01). With mAChR and a-receptor blocked byAtropine and Tolazoline respectively, ACh and NE remain to promote the growth ofcell processes, and the two groups have significant difference compared with thecontrol groups (P＜0.05). The nuclear-cytoplasmic ratio of ACh and NE groups havesignificant difference compared with the control groups (P＜0.05). With receptorsblocked in ACh and NE groups, the nuclear-cytoplasmic ratio increases a little but does not have significant difference (P＞0.05).
3.2.2 The effect of ACh and NE on the cell proliferation activity of EC9706, theesophageal carcinoma cell line in vitro
Both ACh and NE can degrade the cell proliferation activity of EC in vitro, andhave significant difference compared with the control groups (P＜0.05), and can stilldegrade the cell proliferation activity of EC in vitro with mAChR and a-receptorblocked.
3.2.3 The effect of ACh and NE on the cdi proliferation inhibition ratio ofEC9706, the esophageal carcinoma cdl line in vitro
ACh and NE can elevate the cell proliferation inhibition ratio of EC9706 theesophageal carcinoma cell line in vitro, and have the effect of restraining the cellproliferation. The groups have significant difference compared with the controlgroups (P＜0.05), and the inhibition ratio of the colony formation descends withreceptors blocked Atropine and Tolazoline.
3.2.4 The effect of ACh and NE on the AChR, CHAT, PRX1, Annexin-2, Keratin5and K20 expression of EC9706 the esophageal carcinoma cell line in vitro
ACh and NE can obviously promote the AChR, CHAT, PRX1, Annexin-2,Keratin5 and K20 expression of EC9706 the esophageal carcinoma cell line in vitro,and the two groups have significant difference compared with the control groups (P＜0.05). And they can still promote the functional proteins expression of theesophageal carcinoma cell in vitro with mAChR and a-receptor blocked; moreoverwith mAChR blocked by atropine, the ACh groups still have significant differencecompared with the control groups (P＜0.05).
3.2.5 The effect of ACh and NE on the cell cycle and apoptosis of EC9706 theesophageal carcinoma cell line in vitro
ACh and NE can block the cell cycle, making the cells stopped in G1 phase andG2 phase, and reduce the carcinoma cell population in S phase compared with thecontrol groups. The blockage effect is in a descending order: ACh+NE＞NE+Tolazoline＞NE＞ACh+Atropine＞ACh, and the blockage ratio is respectively 90％, 70％, 15％, 15％, 10％compared with the control groups. ACh and NE caninduce the apoptosis of the esophageal carcinoma cell in vitro, and the apoptosis ratioof the carcinoma cells induced is respectively 30 times(ACh group), 4 times (ACh+Atropine group), 10 times (NE group), 20 times (NE+Tolazoline group), and 30times (ACh+NE group) that of the control group.
3.2.6 The effect of ACh and NE on the Annexin-2 mRNA expression of EC9706the esophageal carcinoma cell line in vitro
ACh and NE can promote the multiplication of Annexin-2 mRNA expression inEC9706 the esophageal carcinoma cell line in vitro, and the two groups havesignificant difference compared with the control groups (P＜0.05). The Annexin-2mRNA expression level of ACh group, ACh+Atropine group, ACh+NE group, NEgroup and NE+Tolazoline group is respectively 20 times, 8 times (NE group), 3 times,2 times, and 4 times higher than that of the control group.
3.3 The effect of ACh and NE on the proliferation and differentiation of EC9706cells with ERK1/2 blocked
As ERK1/2 blocked, there arc some changes with the morphous of EC9706 theesophageal carcinoma cell line in vitro, the processes becoming short andnuclear-cytoplasmic ratio augmenting, and that group has significant differencecompared with the control groups (P＜0.05). With ERK1/2 blocked, ACh and NEdecrease the nuclear-cytoplasmic ratio, which is significant statistically (P＜0.05),and the short processes grow a little longer, which is not significant statistically (P＞0.05). With ERK1/2 blocked, the expression of AChR, CHAT, Anncxin-2 and Keratin20 in EC9706 the esophageal carcinoma cell strain in vitro decreases significantly (P＜0.05), while in the groups added ACh and NE, the descended expression increasesrelatively and that is significant statistically (P＜0.05). With ERK1/2 blocked, the cellproliferation activity in EC9706 the esophageal carcinoma cell line in vitro decreasessignificantly (P＜0.05) and the cell proliferation inhibition ratio increasessignificantly, while in the groups added ACh and NE, the effect is further enhanced.With ERK1/2 blocked in EC9706 the esophageal carcinoma cell line in vitro, the cellsare stopped in S phase with the population increasing and percentage of apoptosis cells decreased, while ACh and NE can further reduce the cell percentage resultingfrom ERK1/2 blocked in S phrase (period of cell cycle). The intensity of blockageeffect ranks in a descending order: ACh+NE＞ACh+A＞ACh＞NE+Tolazoline＞NE.
4 CONCLUSIONS
4.1 During the process of carcinogenesis development, the esophageal intramuralnerves decreased gradually or disappeared and the expression of AChR, CHAT,Annexin-2, PRX1 and K20 reduced gradually as well. These findings indicate that thedecrease or dysfunction of intramural nerves may be the nervous factors of theesophageal carcinogenesis. With the generation and development of carcinogenesis,the change pattern of AChR is that the expression decreases gradually in cellmembrane and cytoplasm at first, and then shifts or concentrates from cytoplasm intonuclear, which is possibly the early receptor event that reduces sensitivity of epithelialcells in esophageal mucosa to nervous ACh and furthermore leads to the priming ofcarcinogenesis. The elevation of PRX1 might mean the active molecules that protectthe cells stimulated by injuries keep Annexin-2 and Keratin20 to express, while thedecrease of PRX1 might be the internal cause that leads to the descent ofself-protecting and self-stabilizing function and promotes the carcinogenesis andmalignant transformation.
4.2 ACh and NE could induce cell differentiation of EC9706 (the esophagealcarcinoma cell line) in vitro, and still could promote these cells differentiation afterthe mAChR and a receptor were blocked with Atropine and Tolazoline respectively.These findings demonstrate that the esophageal carcinoma cell could be induced orincreased its ability to differentiate, making the soma augment, the processes eruptand the nuclear-cytoplasmic ratio descend, and the expression of functional proteinsincrease. The mechanism may be through nAChR andβreceptor.
4.3 Both ACh and NE can degrade the cell proliferation activity of EC in vitro. Itappears that they induce carcinoma cell converted from S phase to G1 phase and G2phase, or block the cycle and affect the cell proliferation. This effect is regulated bymulti-receptor and multi-path.
4.4 ACh and NE can promote the multiplication of Annexin-2 mRNA expression inEC9706 the esophageal carcinoma cell line in vitro. ACh has the most powerful effectand augments the expression by 20 times, and ACh +Atropine 8 times, NE,NE+Tolazoline and ACh+NE has feeble effect and can only increase the expressionby 2～4 times.
4.5 ERK1/2 blocker degrades the cell proliferation vigor as well as restrains the celldifferentiation. ACh and NE enhance the degradation of the cell proliferation vigor ofEC9706 the esophageal carcinoma cell line in vitro, with ERK1/2 blocked, and theyinduce the cell differentiation by regulating the functional proteins expression ofcarcinoma cell. The discovery of the mutual relationship of the neurotransmitter andthe ECM blocker will provide an original research idea and direction for furtherrevealing the molecular mechanism of EC generation and development in vivo, andprovide new targets for the drug design used on prevention and cure of the esophagealcarcinoma.
河南林州(原林县)及其毗邻的地区是世界上食管癌(esophagaeal cancer，EC)发病率和死亡率最高的地区，而且，EC当前仍是该地区肿瘤相关疾病的主要死亡原因。但是，目前食管癌变的分子机制仍不十分清楚。最近有文献报道食管癌的发生发展与个体的心理应激、神经-免疫因素等密切相关。
神经系统可经中枢—周围自主神经(交感神经和迷走神经等)通路调节内脏效应细胞。有研究报道，食管闭锁患者迷走神经和喉返神经发育不良；动物实验证实，去除颈段神经嵴或颈交感神经节均可导致食管黏膜缺如和食管闭锁，表明食管与其神经之间在结构和功能上存在着密切关系。近年的临床研究发现，食管癌切除术保留迷走神经，可使患者术后恢复加快，肿瘤复发延迟。西方学者进行大量的研究认为，食管运动功能紊乱所致胃．食管反流可能是导致食管腺癌发生的危险因素之一。这些研究提示，迷走神经功能紊乱或缺陷可能在食管癌变中起着重要的作用。
迷走神经的副交感纤维和交感神经通过释放乙酰胆碱(Acetylcholine，ACh)和去甲肾上腺素(Norepinephrine，NE)及其受体(Acetylcholine receptor，AChRs)实现对食管的功能调节。神经生物学研究发现AChRs对细胞增殖、分化、细胞骨架形成具有重要的生理作用，其激动剂尼古丁可促进胶质细胞肿瘤的细胞增殖，而拮抗剂筒箭毒则具有抑制细胞增殖的效应。已有临床调查提示，恶性肿瘤发生与mAChR功能异常有关。还有报道NE可调节中枢神经元的分化类型，亦可诱导人的肺泡细胞系凋亡。但有关神经及其递质对食管癌变的影响，尚未见报道。我们认为食管癌变及其演进是通过多因素、多阶段、多基因的作用，且具有复杂的机制。本实验室多年的研究也提示多个抑癌基因和癌基因以及蛋白质的功能紊乱是导致食管癌变始动的原因，但尚未能发现关键的基因与蛋白分子。食管癌变的好发部位为食管中下段，食管癌前病变并具有双向发展的不稳定特性，提示食管癌变具有可逆性。另有研究发现激活脑干5-HT中缝核—迷走神经通路对内脏具有保护作用，表明中缝核5-HT神经元通过释放5-HT调节着迷走神经的功能。有关心理应激(Stress)的研究发现，长期的心理压力或抑郁可导致脑内5-HT及其受体水平降低，提示应激可影响迷走神经至食管的胆碱能功能下降，进而推测食管病理变化可能与迷走神经至食管神经通路的结构和功能异常有关，而且食管神经功能异常在食管癌变与食管癌细胞恶性转化中占有重要地位。近年的研究提示，EC细胞角质蛋白(Keratin)、氧化还原酶(PRX1)、钙依赖磷脂结合蛋白(Annexin-2)表达异常。Keratin 5和Keratin 20分别为上皮祖细胞和成熟细胞的标志，也是上皮细胞起黏附、支持和保护的功能基础。PRX1对细胞DNA损伤具有修复作用。Annexin-2具有调节细胞分化和抑制细胞增殖的作用等，可能是抑制食管癌变的重要分子之一。AChR和胆碱乙酰基转移酶(CHAT)是ACh作用于细胞的传递分子。微管相关蛋白(MAP2)是神经元的骨架连接蛋白，退行性变的神经元及神经纤维表达减少。上述分子为进一步研究提供了重要的线索和观察的指标。
因此，本研究采用组织切片、HE染色、免疫荧光、免疫组化染色等方法对食管癌壁间神经丛的形态与食管癌细胞功能基因与蛋白表达变化进行观察，对其与食管癌变和癌细胞转化的关系予以分析。并进一步采用体外细胞培养法、免疫荧光与免疫组化染色、流式细胞以及RT-PCR技术等探讨交感和副交感神经及其递质NE与ACh对食管癌细胞抑癌基因和功能蛋白表达变化的影响，分析ACh与NE对食管癌变与癌细胞转化的作用，旨在揭示与阐明食管神经及其递质变化在食管癌变中的作用和意义，并为食管癌的靶向防治提供新的研究思路。
2材料与方法
2．1 EC壁间神经形态与癌细胞功能蛋白表达变化关系的观察
42例食管癌标本均来自河南省林州市中心医院和姚村食管癌医院，男性37例，女性5例，其平均年龄为60.3±6.5。所有患者在术前均未接受任何化疗或放疗。经4％的多聚甲醛固定、石蜡包埋、连续切片，采用免疫荧光、免疫组化ABC法，对癌、癌旁和正常组织进行免疫组化染色，观察与分析癌、癌旁和正常组织壁间神经形态与癌细胞AChR、ChAT、Annexin-2、PRX1、MAP2、Keratin5和Keratin20表达变化之间以及各功能蛋白表达变化相互之间的关系，以了解食管神经在食管癌变过程中的作用及其变化特点。
2．2体外细胞培养实验
采用细胞培养法，并借助细胞受体阻断剂(阿托品、托拉唑林)、细胞外基质阻断剂(U0126)对人食管癌细胞株EC9706细胞(郑州大学医学院癌症研究室所保存食管鳞癌细胞株)进行体外培养，应用免疫组化、免疫荧光、MTT、平板细胞克隆、流式细胞、荧光定量RT-PCR等技术方法，观察ACh、NE处理前后EC9706细胞的AChR、ChAT、Annexin-2、Keratin 5和Keratin 20等表达以及细胞增殖周期与增殖活力的变化，分析ACh、NE以及细胞外基质对食管癌细胞增殖和分化的影响与相互关系，以揭示神经递质对食管癌变、癌细胞转化的作用及其可能性分子机制。
2．3统计学分析
实验数据采用SPSS10.0统计学软件处理，选用单因素方差分析和配对样本t检验。数据资料用(?)±s表示，以a-0.05为检验水准，P＜0.05表示差异有显著意义，P＜0.01表示差异有非常显著意义。
3结果
3．1食管壁间神经形态与功能蛋白表达的变化及其关系
食管壁间神经MAP2阳性强度由正常到增生、原位癌、癌逐渐减淡，细纤维减少至丧失。AChR、ChAT、Annexin-2、PRX1、K20表达随癌变演进和食管壁间神经退行变的加重逐步减少，Keratin 5在癌变过程中变化较小。Keratin 20、Annexin-2的表达变化与PRX1表达一致。AChR表达变化的特征是随癌变的演进开始由胞膜、胞浆随即逐步减少，而后从胞质向核内集中或转移。ChAT在癌变及其恶性转化过程中变化与减少较晚，非典型增生阶段呈现过表达现象。
3.2 ACh与NE对人食管癌细胞系细胞增殖、分化的影响
3．2．1 ACh和NE对体外培养食管癌细胞系EC9706细胞生长形态的影响
应用ACh和NE培养后，可促使细胞体增大，突起长出，核质比下降；NE可使体外培养的食管癌细胞密度降低，细胞突起更长。ACh和NE两组的突起长度与其余各组相比均具有非常显著性差异(P＜0.01)。在mAChR和a受体阻断后，ACh和NE仍具有促进细胞突起生长的作用，两组与对照组相比具有显著性差异(P＜0.05)。ACh和NE组核质比与对照组相比具有显著性差异(P＜0.05)；ACh、NE组受体阻断前、后相比，核质比有所增大，但无显著性差异(P＞0.05)。
3．2．2 ACh和NE对体外培养食管癌细胞系EC9706细胞增殖活性的影响
ACh和NE均具有降低体外培养食管癌细胞增殖活性的作用，与对照组相比具有显著性差异(P＜0.05)；mAChR和a受体阻断后，仍保留有部分降低体外培养食管癌细胞增殖活力的作用。
3．2．3 ACh和NE对体外培养食管癌细胞系EC9706细胞增殖抑制率的的影响
ACh、NE可提高体外培养食管癌细胞系EC9706细胞增殖抑制率，对细胞增殖具有抑制作用，与对照组相比具有差异显著性(P＜0.05)。受体阻断后，集落形成抑制率仍下降。
3．2．4 ACh和NE对体外培养食管癌细胞系EC9706细胞AChR、ChAT、PRX1、Annexin-2、Keratin5和K20表达的影响
ACh和NE可促进体外培养食管癌细胞系EC9706细胞的AChR、ChAT、PRX1、Annexin-2、Keratin5和K20表达明显增多，与对照组相比具有显著性差异(P＜0.05)；在mAChR和a受体阻断后，仍保留有部分促进体外培养食管癌细胞功能蛋白表达的作用，其中ACh经阿托品阻断mAChR后，与对照组相比，仍较对照组为高，具有统计学意义(P＜0.05)。
3．2．5 ACh和NE对体外培养食管癌细胞系EC9706细胞周期与凋亡的影响
ACh和NE对细胞周期具有阻滞作用，可使细胞阻滞于G1期或G2期，与对照组相比可使S期癌细胞数量减少，阻滞作用由强到弱，依次为：ACh+NE＞NE加托拉唑林＞NE＞ACh加阿托品＞ACh，阻滞率分别占对照组的90％、70％、15％、15％、10％。ACh和NE具有诱导体外培养食管癌细胞凋亡的作用，诱导癌细胞凋亡率分别是对照组的30倍(ACh)、4倍(ACh加阿托品)、10倍(NE)、20倍(NE+托拉唑啉)、30倍(ACh+NE)。
3．2．6 ACh和NE对食管癌细胞系EC9706细胞Annexin-2 mRNA表达的影响
ACh和NE可促使体外培养食管癌细胞系EC9706细胞Annexin-2 mRNA表达增多，与对照组相比具有显著性差异(P＜0.05)；ACh、ACh+阿托品、ACh+NE、NE、NE+T的Annexin-2 mRNA表达水平分别比对照组增高20倍、8倍、3倍、2倍、和4倍。
3．3 ACh和NE对阻断ERK1／2食管癌细胞系EC9706细胞增殖和分化的影响
阻断ERK1／2体外培养食管癌细胞系EC9706细胞形态的突起变短，核质比增大，与对照组相比均有显著差异(P＜0.05)。ACh和NE可使ERK1／2阻断后细胞核质比下降，具有统计学意义(P＜0.05)；变短的突起稍长，但无显著差异(P＞0.05)。阻断ERK1／2体外培养食管癌细胞系EC9706细胞的AChR、ChAT、Annexin-2、Keratin20表达均显著减少(P＜0.05)，加用ACh和NE可使下降的AChR、ChAT、Annexin-2、Keratin20表达增多，具有统计学意义(P＜0.05)。阻断ERK1／2食管癌细胞系EC9706可使细胞的增殖活性下降，细胞增长抑制率提高(P＜0.05)，加用ACh和NE使ERK1／2降低的细胞增殖活性再度下降，细胞增长抑制率再度提高。阻断ERK1／2使体外培养食管癌细胞系EC9706细胞数目在S期阻滞与增多，凋亡细胞百分数减少，ACh和NE可使由ERK1／2阻断引起的S期细胞百分数降低，其作用由强到弱分别为ACh+NE＞ACh+A＞ACh＞NE+托拉唑林＞NE。
4结论
4．1食管壁间神经纤维在癌变演进过程中逐渐减少或丧失，AChR、ChAT、Annexin-2、PRX1、K20随癌变的表达逐步减少。结果提示，食管壁间神经减少或功能丧失可能是食管癌变的神经因素。AChR随癌变始动与演进的特征性变化是其在胞膜、胞浆的含量减少最早，并从胞质向核内集中或转移，这可能是引起食管黏膜上皮细胞对神经性ACh敏感度下降，进而导致癌变启动较早的受体事件。PRX1表达增多可能是受伤害刺激的细胞维持Annexin-2和Keratin20表达有效分子，其表达下降可能是导致细胞自身保护和稳定功能下降，细胞癌变与癌细胞恶化的内在原因。
4．2ACh和NE具有诱导体外培养的人食管癌细胞系EC9706细胞分化的作用，采用阿托品和妥拉唑啉分别阻断mAChR和α受体，仍具有促进EC9706细胞分化的作用。结果提示，食管癌细胞具有经诱导分化或增强分化的能力，ACh和NE可促使细胞体增大，突起长出，核质比下降，功能蛋白表达增多，其机制可能是主要通过nAChR和β受体。
4．3 ACh和NE对体外培养食管癌细胞系EC9706细胞增殖活性具有降低的作用，表现为将癌细胞由S期向G1和G2期诱导，或使其周期发生阻滞，影响细胞的增殖，该作用经多受体、多途径韵调节。
4．4 ACh和NE对体外培养食管癌细胞系EC9706细胞Annexin-2 mRNA表达具有促进作用。ACh作用最强，可使其表达量增加20倍，ACh+阿托品使之增加8倍，NE、NE+托拉唑啉、ACh+NE作用较弱，仅能使之表达增加2～4倍。
4．5 ERK1／2阻滞剂除具有降低细胞增殖和阻滞细胞分化的作用。ACh和NE对阻断ERK1／2体外培养食管癌细胞系EC9706细胞的增殖活性具有再度降低的作用，并通过调节癌细胞功能蛋白的表达而诱导细胞分化。神经递质与细胞外基质阻断剂相互作用的发现为进一步揭示食管癌在体内发生发展的分子机制提供了新的研究思路和方向，并为食管癌防治的药物设计提供了新的靶点。
1 BACKGROUND
Linzhou City (formerly Linxian County), together with nearby counties in Henan,has been well recognized as the highest morbidity and mortality area for esophagealcancer (EC) in the world. Moreover, EC remains currently the leading cause ofcancer-related death in these areas. Nevertheless, the molecular mechanism ofesophageal carcinogenesis is largely unknown. Recent studies have indicated that thegeneration and development of EC are correlated with the individual's psychologicalstress and neuroimmunologic factors.
The viscera effector cells can be regulated by the nervous system viacenter-peripheral autonomic nervous (sympathetic nerve and vagus nerve) pathway. Ithas been reported that there is hypogenesis of vagus nerve and recurrent laryngealnerve in esophageal atresia patients. And animal experiments also demonstrated thatthe removal of either cervical nervous crest or cervical sympathetic ganglia can leadto esophageal mucosa absence and esophageal atresia, which indicates thatesophageal diseases are closely related to esophageal nerves both in structure and function. Recent clinical researches indicate that EC exsection with vagus nervereserved could accelerate postoperative recovery and procrastinate the tumor relapse.Western scholars have carded out a great deal of researches and presume thatgastroesophageal reflux disease (GERD) resulting from functional disorder ofesophageal motor may contribute to esophageal carcinogenesis, which indicates thatthe functional disorder or defection of vagus nerve may play an important role inesophageal carcinogenesis.
The acetylcholine (ACh) and norepinephrine (NE) released fromparasympathetic fibers of vagus nerve and sympathetic fibers and their receptors carryout the regulation of esophageal function. Neurobiological researches demonstrate thatacetylcholine receptors (AChRs) have significant physiological functions on cell proliferation,differentiation and formation of cytoskeleton. Nicotine, the excitomotor of AChRs, prompts theproliferation of gliacyte tumor while tubocurarine (TC) the antagon inhibits that. Clinicalinvestigations have shown that the generations of malignant tumors were correlatedwith dysfunction of mAChR. And it is also reported that NE can regulate the differential typeof central neuron and induce the apoptosis of alveolar cell. But there is no report about the effectof nerves and corresponding transmitters on esophageal carcinogenesis. We presume thatesophageal carcinogenesis is a multistage progressive process involved by multiple factors,multiple genetic changes and their complicated mechanism. Researches of years in ourlaboratory also indicate that a great deal of anti-oncogenes and oncogenes and the functionaldisorders of proteins are the original causes of esophageal carcinoma, but the key genes andprotein molecules haven't been identified. It is discovered from recent census andfollow-up visits that the predilection sites for esophageal carcinogenesis are the middleand inferior segments, and the character of precancerous lesions are of two-waydevelopment, which indicates that the carcinogenesis is reversible. Other studies which find thatthe activation of the brainstem 5-HT (5-hydroxytryptamine, 5-HT) rapheal nuclei-vagus pathwaycan protect the viscera manifested that the rapheal nuclei 5-HT neuron regulates the vagus nerveby releasing 5-HT. Researches on psychological stress discovered that long-standing mental stressor depression leads to descending of center 5-HT and its receptor level, indicating that stress maydegrade the cholinergic function from vagus nerve to esophagus, thus it can be inferred that the esophageal pathological changes may concern the structural and functional abnormality of theneural pathway from vagus nerve to esophagus, and that the disfunction of the esophageal nervetakes a significant place in esophageal cancerization and the malignant transformation ofesophageal carcinoma cells. Recent studies have indicated that EC cell has the feature ofabnormally expressing Keratins, Peroxiredoxin-1 (PRX1) and calcium-dependent lipid bindingprotein-2 (Annexin-2). Keratin 5 (KS) and Keratin 20 (K20) are the marks of progenitorepithelium cells and mature cells respectively, and they are also the foundation of epithelium celladhering, sustaining and protecting functions. PRX1 has repairing function to damaged DNA;Annexin-2 can suppress cells' proliferation and regulate cells' differentiation and it may be one ofthe important molecules inhibiting esophageal carcinogenesis. AChR and Cholineacetyltransferase (CHAT) are transmitting molecules by which ACh acts on cells.Microtubule-associated protein-2 (MAP2) is a kind of skeleton connection protein in the neuron,the expression of which may be decreased in degenerating neurons and nerve fibers.These molecules mentioned here are provided as observational indexes for furtherresearch.
Therefore, the design of this research adopts the methods and techniques of tissues sliced, HEstraining, immunofluorescence (IMF), immunohistochemical straining and so on to survey thedistribution features of esophageal nerves, the intramural neuroplexus morphous of the esophagealcarcinoma, and the changes of the functional genes and protein expression, and to analyze therelationship between those indexes mentioned above and the carcinogenesis and celltransformation.Furthermore, we use vitro cell culture, immunofluorescence, immunohistochemicalstraining, flow cytometry, RT-PCR etc. to make an exploratory study on the affection of NE, thesympathetic transmitter, and ACh, the parasympathetic transmitter, on the changes inanti-oncogenes and functional protein expression of the esophageal carcinoma, thus we cananalyze the effects of NE and ACh on esophageal carcinogenesis and cell transformation, which isof significance in revealing and illuminating the molecular mechanism of esophagealcarcinogenesis, and providing new research ideas on target for cancer prevention and treatment.
2 MATERIAL and METHODS
2.1 Observation of the relationship between EC intramural nerve morphous and expression changes of cancer cellular functional proteins.
There were 42 EC specimens that were all from Central Hospital in Linzhou orYaocun EC Hospital and none of the patients had accepted any chemotherapy orradiotherapy. The 42 patients included 37 males and 5 females, whose mean age was60.3±6.5. After 4% polyformalin fixation, paraffin imbedding, serial sectioning anddying the cancer tissue, para (adjacent) carcinoma tissue and normal tissue via IMFand immunohistochemical ABC method, we surveyed and analyzed the relationsamong intramural nerve morphous of the three kinds of tissues and AChR, CHAT,Annexin-2, PRX1, MAP2, Keratin 5 and Keratin 20 expression changes of the cancercells, including relations among expression changes of every functional proteins, so asto identify the change pattern and the effect of esophageal nerves in the process ofesophageal carcinogenesis.
2.2 Experiment of cell culture in vitro
By adding cell receptor blocking agents Atropine and Tolazoline and xtracellularmatrix (ECM) blocking agent U0126, we cultured in vitro the human esophagealcarcinoma cell strain EC9706, the ESCC (esophageal squamous carcinoma cell,ESCC) strain preserved in the Cancer Lab of the Medical College of ZhengzhouUniversity. By means of immunohistochemical straining, IMF, flat cell clone, flowcytometry, fluorescently quantitative RT-PCR and so on, we observed the changes ofthe cell cycle and vigor of cell generation as well as the changes of the expression ofAChR, CHAT, Annexin-2, Keratin 5 and Keratin 20 in EC9706 both before and afterthe treatment of ACh and NE, then analyzed the affection of ACh, NE and ECM to theproliferation and differentiation of EC cells and their mutual relationship, so as todetect the effect of neurotransmittcr in the process of esophageal carcinogenesis andcancer cell transformation and find the possible molecular mechanism.
2.3 Statistics analysis
The data is analyzed with SPSS10.0 by means of one-factor analysis of varianceand paired sample t-test. It is described as (?)±s, with a=0.05 as the size of test andP＜0.05 meaning the variance to be significant.
3 RESULTS
3.1 The relation between esophageal intramural nerve morphous and expressionchanges of cancer cellular functional proteins
The MAP2 positive intensity of esophageal intramural nerve decreases graduallyin the order of normal tissue, hyperplasia, preinvasive carcinoma and cancer, whilethe fine nerve fibers reduce and finally disappear. The expressions of AChR, CHAT,Annexin-2, PRX1, and Keratin 20 decrease gradually along with the development ofcarcinogenesis and the aggravation of esophageal intramural nerve retrogradation,while Keratin5 changes less in the process of carcinogenesis. The change ofKeratin20、Annexin-2 expression keeps the same with PRX1. The expression ofAChR is typically to decrease gradually from cell membrane to cytoplasm and thenshift or concentrate from cytoplasm into nuclear with the development ofcarcinogenesis. ChAT changes and decreases late in the process of carcinogenesis" andmalignant transformation and expresses excessively in the atypical hyperplasia phase.
3.2 The effect of ACh and NE on the cell proliferation and differentiation ofEC9706, the human esophageal carcinoma cell line cultured in vitro
3.2.1 The effect of ACh and NE on the cell proliferation of EC9706, theesophageal carcinoma cell line in vitro
After treatment of ACh and NE, the cell soma was augmented, the processeserupted and the nuclear-cytoplasmic ratio was decrease. The esophageal carcinomacells grow with lower density and have longer processes under the role of NE in vitro.The process length of ACh and NE groups have highly significant differencecompared with the other groups (P＜0.01). With mAChR and a-receptor blocked byAtropine and Tolazoline respectively, ACh and NE remain to promote the growth ofcell processes, and the two groups have significant difference compared with thecontrol groups (P＜0.05). The nuclear-cytoplasmic ratio of ACh and NE groups havesignificant difference compared with the control groups (P＜0.05). With receptorsblocked in ACh and NE groups, the nuclear-cytoplasmic ratio increases a little but does not have significant difference (P＞0.05).
3.2.2 The effect of ACh and NE on the cell proliferation activity of EC9706, theesophageal carcinoma cell line in vitro
Both ACh and NE can degrade the cell proliferation activity of EC in vitro, andhave significant difference compared with the control groups (P＜0.05), and can stilldegrade the cell proliferation activity of EC in vitro with mAChR and a-receptorblocked.
3.2.3 The effect of ACh and NE on the cdi proliferation inhibition ratio ofEC9706, the esophageal carcinoma cdl line in vitro
ACh and NE can elevate the cell proliferation inhibition ratio of EC9706 theesophageal carcinoma cell line in vitro, and have the effect of restraining the cellproliferation. The groups have significant difference compared with the controlgroups (P＜0.05), and the inhibition ratio of the colony formation descends withreceptors blocked Atropine and Tolazoline.
3.2.4 The effect of ACh and NE on the AChR, CHAT, PRX1, Annexin-2, Keratin5and K20 expression of EC9706 the esophageal carcinoma cell line in vitro
ACh and NE can obviously promote the AChR, CHAT, PRX1, Annexin-2,Keratin5 and K20 expression of EC9706 the esophageal carcinoma cell line in vitro,and the two groups have significant difference compared with the control groups (P＜0.05). And they can still promote the functional proteins expression of theesophageal carcinoma cell in vitro with mAChR and a-receptor blocked; moreoverwith mAChR blocked by atropine, the ACh groups still have significant differencecompared with the control groups (P＜0.05).
3.2.5 The effect of ACh and NE on the cell cycle and apoptosis of EC9706 theesophageal carcinoma cell line in vitro
ACh and NE can block the cell cycle, making the cells stopped in G1 phase andG2 phase, and reduce the carcinoma cell population in S phase compared with thecontrol groups. The blockage effect is in a descending order: ACh+NE＞NE+Tolazoline＞NE＞ACh+Atropine＞ACh, and the blockage ratio is respectively 90％, 70％, 15％, 15％, 10％compared with the control groups. ACh and NE caninduce the apoptosis of the esophageal carcinoma cell in vitro, and the apoptosis ratioof the carcinoma cells induced is respectively 30 times(ACh group), 4 times (ACh+Atropine group), 10 times (NE group), 20 times (NE+Tolazoline group), and 30times (ACh+NE group) that of the control group.
3.2.6 The effect of ACh and NE on the Annexin-2 mRNA expression of EC9706the esophageal carcinoma cell line in vitro
ACh and NE can promote the multiplication of Annexin-2 mRNA expression inEC9706 the esophageal carcinoma cell line in vitro, and the two groups havesignificant difference compared with the control groups (P＜0.05). The Annexin-2mRNA expression level of ACh group, ACh+Atropine group, ACh+NE group, NEgroup and NE+Tolazoline group is respectively 20 times, 8 times (NE group), 3 times,2 times, and 4 times higher than that of the control group.
3.3 The effect of ACh and NE on the proliferation and differentiation of EC9706cells with ERK1/2 blocked
As ERK1/2 blocked, there arc some changes with the morphous of EC9706 theesophageal carcinoma cell line in vitro, the processes becoming short andnuclear-cytoplasmic ratio augmenting, and that group has significant differencecompared with the control groups (P＜0.05). With ERK1/2 blocked, ACh and NEdecrease the nuclear-cytoplasmic ratio, which is significant statistically (P＜0.05),and the short processes grow a little longer, which is not significant statistically (P＞0.05). With ERK1/2 blocked, the expression of AChR, CHAT, Anncxin-2 and Keratin20 in EC9706 the esophageal carcinoma cell strain in vitro decreases significantly (P＜0.05), while in the groups added ACh and NE, the descended expression increasesrelatively and that is significant statistically (P＜0.05). With ERK1/2 blocked, the cellproliferation activity in EC9706 the esophageal carcinoma cell line in vitro decreasessignificantly (P＜0.05) and the cell proliferation inhibition ratio increasessignificantly, while in the groups added ACh and NE, the effect is further enhanced.With ERK1/2 blocked in EC9706 the esophageal carcinoma cell line in vitro, the cellsare stopped in S phase with the population increasing and percentage of apoptosis cells decreased, while ACh and NE can further reduce the cell percentage resultingfrom ERK1/2 blocked in S phrase (period of cell cycle). The intensity of blockageeffect ranks in a descending order: ACh+NE＞ACh+A＞ACh＞NE+Tolazoline＞NE.
4 CONCLUSIONS
4.1 During the process of carcinogenesis development, the esophageal intramuralnerves decreased gradually or disappeared and the expression of AChR, CHAT,Annexin-2, PRX1 and K20 reduced gradually as well. These findings indicate that thedecrease or dysfunction of intramural nerves may be the nervous factors of theesophageal carcinogenesis. With the generation and development of carcinogenesis,the change pattern of AChR is that the expression decreases gradually in cellmembrane and cytoplasm at first, and then shifts or concentrates from cytoplasm intonuclear, which is possibly the early receptor event that reduces sensitivity of epithelialcells in esophageal mucosa to nervous ACh and furthermore leads to the priming ofcarcinogenesis. The elevation of PRX1 might mean the active molecules that protectthe cells stimulated by injuries keep Annexin-2 and Keratin20 to express, while thedecrease of PRX1 might be the internal cause that leads to the descent ofself-protecting and self-stabilizing function and promotes the carcinogenesis andmalignant transformation.
4.2 ACh and NE could induce cell differentiation of EC9706 (the esophagealcarcinoma cell line) in vitro, and still could promote these cells differentiation afterthe mAChR and a receptor were blocked with Atropine and Tolazoline respectively.These findings demonstrate that the esophageal carcinoma cell could be induced orincreased its ability to differentiate, making the soma augment, the processes eruptand the nuclear-cytoplasmic ratio descend, and the expression of functional proteinsincrease. The mechanism may be through nAChR andβreceptor.
4.3 Both ACh and NE can degrade the cell proliferation activity of EC in vitro. Itappears that they induce carcinoma cell converted from S phase to G1 phase and G2phase, or block the cycle and affect the cell proliferation. This effect is regulated bymulti-receptor and multi-path.
4.4 ACh and NE can promote the multiplication of Annexin-2 mRNA expression inEC9706 the esophageal carcinoma cell line in vitro. ACh has the most powerful effectand augments the expression by 20 times, and ACh +Atropine 8 times, NE,NE+Tolazoline and ACh+NE has feeble effect and can only increase the expressionby 2～4 times.
4.5 ERK1/2 blocker degrades the cell proliferation vigor as well as restrains the celldifferentiation. ACh and NE enhance the degradation of the cell proliferation vigor ofEC9706 the esophageal carcinoma cell line in vitro, with ERK1/2 blocked, and theyinduce the cell differentiation by regulating the functional proteins expression ofcarcinoma cell. The discovery of the mutual relationship of the neurotransmitter andthe ECM blocker will provide an original research idea and direction for furtherrevealing the molecular mechanism of EC generation and development in vivo, andprovide new targets for the drug design used on prevention and cure of the esophagealcarcinoma.
引文
1．胡盛平，杨红珊．食管癌病因学研究的回顾与展望．中国癌症杂志，2001，11：171-173
2．袁媛，张卫东，郗园林．生活事件与食管癌发生的病例对照研究．河南医科大学学报，2001，36：74-76
3．周春锋，夏全，李林海．安县食管癌危险因素的病例对照研究．江苏预防医学，1999，10：14-15
4．俞鲁谊．社会心理与癌症：性格情绪与癌症。中国肿瘤，1992，：27-29
5. Qubain SW, Natsugoe S, Matsumoto S, et al. Micrometastases in the cervical lymph nodes in esophageal squamous cell carcinoma. Dis Esophagus, 2001, 14: 143-148
6. Wang LD, Zhou Q, Feng CW, et al. Intervention and follow-up on human esophageal precancerous lesions in Henan, northern China, a high-incidence area for esophageal cancer. Gan To Kagaku Ryoho, 2002, 29:159-172
7．李秀香，刘广庆，周刚．食管癌病人精神状态异常与脑电图变化．现代电生理学杂志，2000，12：227-228
8. Oliveira RC, Oliveira R, Ferreira CM, et al. Involvement of 5-HT(2) serotonergic receptors of the nucleus raphe magnus and nucleus reticularis gigantocellularis/paragigantocellularis complex neural networks in the antinociceptive phenomenon that follows the post-ictal immobility syndrome. Exp Neurol, 2006, 201:144-153
9. Sampliner RE, Spechler SJ. Controversies in barrett,s esophagus and barret, scancer. World organization for specialized studies on diseases of the esophagus in 8th world congress, 2006, September 3-6
10. Kessler W, Traeger T, Westerholt A, et al. The vagal nerve as a link between the nervous and immune system in the instance of polymicrobial sepsis. Langenbecks Arch Surg, 2006, 391:83-87
11. Guarini S, Altavilla D, Cainazzo MM, et al. Efferent vagal fibre stimulation blunts nuclear factor JB activation and protects against hypovolemic hemorrhagic shock. Circulation, 2003, 107:1189-1194
12．崔忠厚，孙玉鹗，黄孝迈，等．25例电视胸腔镜手术的初步结果．中华外科杂志，1994，32：584-586
13. Balaji NS, Crookes PF, Banki F, et al. A safe and noninvasive test for vagal integrity revisited. Arch Surg, 2002, 137:954-959
14. Yamamoto S, Kawahara K, Maekawa T, et al. Minimally invasive esophagectomy for stage Ⅰ and Ⅱ esophageal cancer. Ann Thorac Surg, 2005, 80: 2070-2075
15．杜贾军，孟龙，陈景寒，等．手辅助电视胸腔镜行食管癌切除术．中华外科杂志，2005，43：351-353
16．王国范，张百江，杨文锋，等．保留迷走神经加胃底重建预防食管贲门癌术后胃食管返流的研究．肿瘤防治杂志，2005，12：218-221
17．蒋跃光．食管疾病．重庆出版社(重庆)，1998：85-86
18. Christian Often, Lucia Migliazza, Huimin Xia, et al. Neural crest-derived defects in experimental esophageal atresia. Pediatr Res, 2000, 47:178-183
19. Qi BQ, Merei J, Hasthorpe S, et al. The vagus and recurrent laryngeal nerves in the rodent experimental model of esophageal atresia. J Pedia Surg, 1977, 32:1580-1586
20．李建国，彭周全，杜朝晖，等．电针足三里激活胆碱能抗炎通路抗大鼠失血性休克的研究．中国中西医结合急救杂志，2006，13：27-31
21. Nordin C, Sjodin I. CSF monoamine patterns in pathological gamblers and healthy controls. J Psychiatr Res. 2006, 40:454-459
22．吕保来，吴爱群，曹莉，等．慢性不可预见性应激对大鼠皮质及海马酪氨酸硝基化的影响解剖学杂志，2006，29：473-475
23. Jeffrey H, Shlley M, Sidney H, et al. Dysfunctional attitudes and 5-HT2A receptors during depression self-harm. Am J Psychiatry, 2003, 160:90-99
24. 24.. Sanchez C, Diaz NJ, Avila J. Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function. Prog Neurobiol, 2000, 61:133-148
25. Richard B, Presland, Richard J. Making sense of the epithelial barrier: what molecular biology and genetics tell us about the functions of oral mucosal and epidermal tissues. Transfer of Advances in Sciences into Dental Education. J Dent Edu, 2002, 66:564-574
26. David JP, Beverly AD, Richard BP. Functional Analysis of the Profilaggrin N-Terminal Peptide: Identification of Domains that Regulate Nuclear and Cytoplasmic Distribution. Journal of Investigative Dermatology, 2002, 119: 661-669
27. Trmbino S, Cesario A, Margaritora S, et al. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesot helioma cells: role of mitogenactivated protein kinase pathway. Cancer Res, 2004, 64:135-145
28. Takita M, Muramatsu I. Alteration of brain nicotine receptor is induced by immobilization stress and nicotinic in rats.Brain Res, 1995, 681:190-192
29. Christian W, Yasutaka H, Dick R. Acetylcholine Increases Intracellular Ca~(2+) Via Nicotinic Receptors in Cultured PDF-Containing Clock Neurons of Drosophila. J Neurophysiol, 2004, 91:912-923
30. Tourovskaia A, Kosar TF, Folch A. Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin. Biophys J, 2006, 90:2192-2198
31．胡昕，吕建新．乙酞胆碱及其拮抗剂对人SK-N-SH细胞增殖及mAchR 1表达的影响．实验生物学报，2005，38：287-296
32. Kinnula VL, Lehtonen S, Sormunen R, et al. Overexpression of peroxiredoxins Ⅰ, Ⅱ, Ⅲ, Ⅴ, and Ⅵ in malignant mesothelioma. J Pathol, 2002, 196:316-323
33. Yanagawa T, lwasa S, lshii T, et al. Peroxiredoxin Ⅰ expression in oral cancer: a potential new tumor marker. Cancer Lett, 2000, 156:27-35
34．沈传陆，朱俊．Prx 1高表达对人PC3前列腺癌细胞氧化损伤的作用．东南大学学报(医学版)，2005，24：377-380
35. Raynal P, Pollard HB. Annexin: The problem of assessing the biological role for gene family of multifunctional calcium and phospholipid-2 binding proteins. Biochem Biophys Acta, 1994, 1197:63-93
36. Cole SP, Pinkoshi MJ, Bhardwaj G, et al. Elevated expression of annexin Ⅱ in a multidrug resistant small cell lung cancer cell line. Br J Cancer, 1992, 65: 498-502
37. Cole SP, Pinkoshi MJ, Bhardwaj G, et al. Elevated expression of annexin Ⅱ in a multidrug resistant small cell lung cancer cell line. Br J Cancer, 1992, 65: 498-502
38．王立东，郑树，刘宾，等．河南食管癌高发区居民食管色素镜检查及手术标本碘染色结果分析．郑州大学学报医学版，2002，37：730-732
39．李质磬，于桂臣．食管神经支配特点的光、电镜观察．解剖学杂志，1996，19：539-542
40．松尾．消化管 endocrine，paracrine，neucrine．医学，1982，120：110
41. Pollard TD, Peters B, Kirfel J, et al. Complete cytolysis and neonatal lethality in keratin 5 knockout mice reveal its fundamental role in skin integrity and in epidermolysis bullosa simplex. Mol Biol Cell, 2001, 12:1775-1789
42. Strnad P, Windoffer R, Leube R E. Induction of rapid and reversible cytokeratin filament network remodeling by inhibition of tyrosine phosphatases. J Cell Sci, 2002, 115:4133-4148
1． Whittaker VP.Handbuch der experimente llenpharma-kologie Bd.Berlin:Springer,1963:1-39
2． Sastry BVR,Sadavongvivad C.Cholinergic systems in non-nervous tissues.Pharmacol Rev,1979,30:65-132
3． Miyazawa A,Fujiyoshi Y,Unwin N.Structure and gating mechanism of the acetylcholine receptor pore.Nature,2003,423:949-955
4． Wessle RI,Kirkpatrick CJ,Racke K.Non-neuronal acetylcholine,a locally acting molecule widely distributed in biological systems:expression and function in human.Pharmacol Ther,1998,77:9279
5． Klapproth H,Remheimer T,Metzen J,et al.Non-neuronal acetylcholine,a signalling molecule synthesized bysurface cell s of rat and man.Naunyn Schmiedebergs Arch Pharmacol,1997,355:515-523
6． Wessle RI,Kilbinger H,Bttinger F,et al.The non-neuronal cholinegic system in humans:expression,function and pathophysiology.Life Sci,2003,72:2055-2061
7． Trmbino S,Cesario A,Margaritora S,et al.Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesot helioma cells:role of mitogen-activated protein kinase pathway.Cancer Res,2004,64:135-145
8． Takita M,Muramatsu I.Alteration of brain nicotine receptor is induced by immobilization stress and nicotinic in rats.Brain Res,1995,681:190-192
9． Christian W,Yasutaka H,Dick RN.Acetylcholine Increases Intracellular Ca~(2+)Via Nicotinic Receptors in Cultured PDF-Containing Clock Neurons of Drosophila.J Neurophysiol,2004,91:912-923
10． Tourovskaia A,Kosar TF,Folch A.Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin.Biophys J,2006,90:2192-2198
11． Engel AG,Ohno K,Sine SM.Congenital myasthenic syndromes:experiments of nature.J Physiol Paris,1998,92:113-117
12．张栩，丛志强，李海峰，等．重症肌无力与胸腺外恶性肿瘤相关性的研究．中华神经科杂志，2005，22：263-265
13. Catherine C, Krishna S, David R, et al. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the β-adrenergic pathway. Circulation. 1998;98:1329-1334
14. Fu Y, Hwang B, Chiu Y, et al. Norepinephrine induces apoptosis in neonatal rat cardiomyocytes through a reactive oxygen species-caspase signaling pathway. Cardiovascular Research, 2004, 62:558-567
15. Dincer HE, Gangopadhyay N, Wang R, et al. Norepinephrine induces alveolar epithelial apoptosis mediated by alpha-, beta-, and angiotensin receptor activation.. Am J Physiol Lung Cell Mol Physiol, 2001, 281:1624-1630
16. Singal T, Dhalla NS, Tappia PS. Norepinephrine-induced changes in gene expression of phospholipase C in cardiomyocytes. J Mol Cell Cardiol, 2006, 41: 126-37
17. Viemari JC, Ramirez JM. Norepinephrine differentially modulates different types of respiratory pacemaker and nonpacemaker neurons. J Neurophysiol, 2006, 95: 2070-82
18．吴爱群，王立东，常志伟，等．乙酰胆碱与去甲肾上腺素对EC109细胞分化的调节作用及其可能性机制．中华肿瘤防治杂志，2007，14：36-39
19．张驰宇，张高红，杨敏，等．四步法消除SYBRGreenl实时定量RT-PCR中引物二聚体的影响．中国生物化学与分子生物学报，2004，20：387-392
20．张蓓．实时荧光定量PCR的研究进展及其应用．国外医学临床生物化学与检验学分册，2003，24：327-329
21．欧阳松应，杨冬，欧阳红生，等．实时荧光定量PCR技术及其应用生命的化学，2004，24：74-76
22. David JP, Beverly AD, Richard BE Functional Analysis of the Profilaggrin N-Terminal Peptide: Identification of Domains that Regulate Nuclear and Cytoplasmic Distribution. Journal of Investigative Dermatology, 2002, 119: 661-669
23. Raynal P, Pollard HB. Annexin: The problem of assessing the biological role for gene family of multifunctional calcium and phospholipid-2 binding proteins. Biochem Biophys Acta, 1994, 1197:63-93
24. Hajjar KA, Krishnan S. Annexin Ⅱ: A mediator of the plasmin/plasminogen activator system. Trends Cardiovasc Med, 1999, 9:128-138
25. Kang HM, Choi KS, Kassam G, et al. Role of annexin Ⅱ tetramer in plasminogen activation. Trends Cardiovasc Med, 1999, 9:92-102
26. Mai JX, Waisman DM, Sloane BF. Cell surface complex of cathepsin B/annexin Ⅱ tetramer in malignant progression. Biochem Biophys Acta 2000, 149:301-322
27. Siever DA, Efickson HP. Extracellular annexin Ⅱ. Int J Biochem Cell Biol, 1997, 29:1219-1223
28. 28. Kinnula VL, Lehtonen S, Sormunen R, et al. Overexpression of peroxiredoxins Ⅰ, Ⅱ, Ⅲ, Ⅴ, and Ⅵ in malignant mesothelioma. J Pathol, 2002, 196:316-323
29．沈传陆，朱俊．Prx 1高表达对人PC3前列腺癌细胞氧化损伤的作用．东南大学学报(医学版)，2005，24：377-380
30. Kumoto K, Sawada H, Yamada Y, et al. Annexin Ⅱ overexpression is correlated with poor prognosis in human gastric carcinoma. Anticancer Res, 2001, 21: 1339-1345
31. Takita M, Muramatsu I. Alteration of brain nicotine receptor is induced by immobilization stress and nicotinic in rats. Brain Res, 1995, 681:190-192
32. David JP, Beverly AD, Richard BP. Functional Analysis of the Profilaggrin N-Terminal Peptide: Identification of Domains that Regulate Nuclear and Cytoplasmic Distribution. J Invest Dermatol, 2002,119:661-669
33. Emoto K, Yamaka Y, Sawada H, et al. Annexin Ⅱ overexpression correlates with stromal tenascin-C overexpression: a prognostic marker in colorectal carcinoma. Cancer, 2001, 92:1419-1426
34. Reeves SA, Chave-Kappel C, Davis R, et al. Developmental regulation of annexin Ⅱ in human brain and expression in high grade glioma. Caner Res, 1992, 52:6871-6876
35. Frohlich M, Motte P, Galvin K, et al. Enhanced expression of the protein-kinase substrate p36 in human hepatocellular carcinoma. Mol Cell Biol, 1990, 10: 3216-3223
36. Cole SP, Pinkoshi MJ, Bhardwaj G, et al. Elevated expression of annexin Ⅱ in a multidrug resistant small cell lung cancer cell line. Br J Cancer, 1992, 65: 498-502
37．李习玲，连小华，张诚，等．Annexin Ⅱ在人表皮角质形成细胞分化中的表达．第四军医大学学报，2004，25：1921-1924
1. 1 H ilger RA, Scheulen M E, Strum berg D. The Ras Raf-M EK-ERK pathway in the treatment of cancer. Onkologie, 2002, 25: 511-518
2. Sherr C J. Principles of tumor suppression. Cell. 2004 Jan 23; 116: 235-46
3．龚小卫，姜勇．丝裂原活化蛋白激酶(MAPK)生物学功能的结构基础．中国生物化学与分子生物学报，2003，19：5-11
4. Duanmu DQ, Li ZH, Wang JZ, et al. MAPK signal transduction pathway: the study progress. J Cell Biol, 2002, 24: 151-154
5. Mueller H, Flury N, Eppenberger CS, et al. Potential prognostic value of mitogen activated protein kinase activity for disease free survival of primary breast cancer patients. Int J Cancer, 2000, 89: 384-388
6．李印 周清华 孙芝琳，等．靶向抑制ERK1／2信号传导通路对人高转移大细胞肺癌细胞株L9981恶性表型的影响．中国肺癌杂志，2005，8：504-509
7. Dincer HE, Gangopadhyay N, Wang R, et al. Norepinephrine (NE) induces apoptosis. Am J Physiol Lung Cell Mol Physiol. 2001, 281: 1624-1630
8. Trmbino S, Cesario A, Margaritora S, et al. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesot helioma cells: role of mitogenactivated protein kinase pathway. Cancer Res, 2004, 64: 135-145
9．吴爱群，王立东，常志伟，等．乙酰胆碱与去甲肾上腺素对食管癌细胞分化的调制作用及其机制．中华肿瘤防治杂志，2007，14：36-39
10. Acqua ML, Smith KE, Gorski JA, et, al. Home EA, Regulation of neuronal PKA signaling through AKAP targeting dynamics. Eur J Cell Biol. 2006, 85: 627-33
11. Pei J J, Braak H, An WL, et al. Up-regulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degeneration in Alzheimer's disease. Brain Res Mol Brain Res. 2002, 30; 109: 45-55
12. Trmbino S, Cesario A, Margadtora S, et al. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesot helioma cells: role of mitogenactivated protein kinase pathway. Cancer Res, 2004, 64: 135-145
13. 13. Wessle RI, Kirkpatrick CJ, Racke K. Non-neuronal acetylcholine, a locally acting molecule widely distributed in biological systems: expression and function in human. Pharmacol Ther, 1998, 77: 923-919
14. Klapproth H, Reinheimer T, Metzen J, et al. Non-neuronal acetylcholine, a signalling molecule synthesized by surface cells of rat and man. Naunyn Schmiedebergs Arch Pharmacol, 1997, 355:515-523
15. 15. Takita M, Muramatsu I. Alteration of brain nicotine receptor is induced by immobilization stress and nicotinic in rats. Brain Res, 1995, 681:1902192.
16. Knobloch O, Tischner R. Characterization of nitrate reductase deficient mutants of Chlorella Sorokini. Physiol,1989, 89:78 6-791
17. 16. Zhang XJ, Yang L, Caen JP, et al. Induction of acelcholinestease expression during apoptosis in various cell types. Cell Dezth Differ, 2002, 9:790～800
18．胡昕，吕建新．乙酞胆碱及其拮抗剂对人SK-N-SH细胞增殖及mAchR 1表达的影响．实验生物学报，2005，38：287～296
19. DeFea K A, Zalevsky J, Thoma MS, et al. β-Arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol, 2000, 148,: 1267-1282
20. Girnita L, Shenoy S K, Sehat B, et al. beta-Arrestin and Mdm2 Mediate IGF-1 Receptor- stimulated ERK Activation and cell cycle progression. J. Biol. Chem. 2007, 282(1): 11329-11338
21. Arora, P., Ricks, T. K., Trejo, J. Protease-activated receptor signalling, endocytic sorting and dysregulation in cancer. J. Cell Sci. 2007, 120:921-928
22. Goetzl EJ. Diverse pathways for nuclear signaling by G protein-coupled receptors and their ligands. FASEB J. 2007, 21:638-642
23. Ma, L., Pei, G. beta-arrestin signaling and regulation of transcription. J. Cell Sci.. 2007 80:1214-1221
24. Kinnula VL, Lehtonen S, Sormunen R, et al. Overexpression of peroxiredoxins Ⅰ, Ⅱ, Ⅲ, Ⅴ, and Ⅵ in malignant mesothelioma. J Pathol, 2002, 196:316-323
25．沈传陆，朱俊．Prx 1高表达对人PC3前列腺癌细胞氧化损伤的作用．东南大学学报(医学版)，2005，24：377-380
26. 26. Richard B, Presland, Richard J. Making sense of the epithelial barrier: what molecular biology and genetics tell us about the functions of oral mucosal and epidermal tissues. J Dent Edu, 2002, 66:564-574
1．林东昕．中国食管癌分子流行病学研究．中华流行病学杂志，2003，24：939-943
2．王立东，郑树．食管癌研究的历史回顾和哲学思考．医学与哲学，2001，22：1-5
3．袁媛，张卫东，郗园林．生活事件与食管癌发生的病例对照研究．河南医科大学学报，2001，36：74-76
4. Wang LD, Zhou Q, Feng CW, et al. Intervention and follow-up on human esophageal precancerous lesions in Henan, northern China, a high-incidence area for esophageal cancer. Gan To Kagaku Ryoho, 2002, 29:159-172
5．李秀香，刘广庆，周刚．食管癌病人精神状态异常与脑电图变化．现代电生理杂志，2000，7：24-25
6. Bonne O, Brandes D, Gilboa A, et al. Longitudinal MRI study of hippocapal volume in trauma survivors with depression. Am J Psychiatry, 2001, 158: 1248-1251
7. Nordin C, Sjodin I. CSF monoamine patterns in pathological gamblers and healthy controls. J-Psychiatr-Res. 2006, 40:454-459
8. Jeffrey H, Shlley M, Sidney H, et al. Dysfunctional attitudes and 5-HT2A receptors during depression self-harm. Am J psychiatry, 2003, 160:90-99
9. Keeney A, Jessop D-S, Harbuz M-S, et al. Differential effects of acute and chronic social defeat stress on hypothalamic-pituitary-adrenal axis function and hippocampal serotonin release in mice. J-Neuroendocrinol. 2006, 18:330-338
10. Ferretti C, Blengio M, Gamalero SR, et al. Biochemical and behaviour changes induced by acute stress in chronic stress model of depression: the effect of amitriptyline. Eur J phanacol. 1995, 280:19-26
11. Sarbaka SN, Saha K. Moderate exercise and chronic stress produce counteractive effects on different areas of the brain by acting through various neurotransmitter receptor subtypes: a hypothesis. Theor-Biol-Med-Model. 2006, 3:33-38
12．吕保来，吴爱群，曹莉，等．慢性不可预见性应激对大鼠皮质及海马酪氨酸硝基化的影响．解剖学杂志，2006，29：473-475
13. Weisstaub NV, Zhou M, Lira A, et al. Cortical 5-HT2A receptor signaling modulates anxiety-like behaviors in mice. Science. 2006, 313:536-540
14. Gilbertson MW, Shenton MW, Ciszeuski A, et al. Smaller hippocampal volume predicts pathologic vulnerability to psychological trauma. Nat Neurosci, 2002, 5: 1242-1247
15．李鹂，涂自良，杜士明，等．氟西汀对实验性抑郁症大鼠抑郁行为及海马神经元再生的影响．医药导报，2006，25：280-282
16. Marcoli M, Cervetto C, Paluzzi P, et al. Nitric oxide-evoked glutamate release and cGMP production in cerebellar slices: control by presynaptic 5-HT1D receptors. Neurochem-Int, 2006, 49:12-19
17. Munoz Castaneda JR, Montilla P, Padillo FJ, et al. Role of serotonin in cerebral oxidative stress in rats. Acta-Neurobiol-Exp-(Wars). 2006, 66:1-6
18. de-Oliveira RC, de-Oliveira R, Ferreira CM, et al. Involvement of 5-HT(2) serotonergic receptors of the nucleus raphe magnus and nucleus reticularis gigantocellulads/ paragigantocellularis complex neural networks in the anti-nociceptive phenomenon that follows the post-ictal immobility syndrome. Exp-Neurol. 2006, 201:144-153
19. Monti JM, Jantos H. Effects of activation and blockade of 5-HT2A/2C receptors in the dorsal raphe nucleus on sleep and waking in the rat. Prog-Neuropsychopharmacol-Biol-Psychiatry. 2006, 30:1189-1195
20. Trmbino S, Cesario A, Margaritora S, et al. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesot helioma cells: role of mitogen-activated protein kinase pathway. Cancer Res, 2004, 64:135-145
21．汤燕平，工建国．乙酞胆碱对人胃粘膜上皮细胞和胃腺癌细胞N受体p亚单位的影响．中国应用生理学杂志，2005，21：457-460
22. Takita M, Muramatsu I. Alteration of brain nicotine receptor sinduced by immobilization stress and nicotinic in rats. BrainRes, 1995, 681:190-192
23. Guarini S, Altavilla D, Cainazzo MM, et al. Efferent vagal fibre stimulation blunts nuclear factor JB activation and protects against hypovolemic hemorrhagic shock. Circulation, 2003, 107:1189-1194
24．余炜伟，王立东，齐义军，等．食管鳞癌组织中p16基因缺失及甲基化检测．郑州大学学报(医学版)，2006，41：282-284
25．魏亚宁，舒青，张素珍．亚硝胺类化合物诱导小鼠食管病变与基因组甲基化的关系研究．科学技术与工程，2006，6：2036-2040
26．张栩，丛志强，李海峰，等．重症肌无力与胸腺外恶性肿瘤相关性的研究．中华神经科杂志，2005，22：263-265
27. Kessler W, Traeger T, Westerholt, A, et al. The vagal nerve as a link between the nervous and immune system in the instance of polymicrobial sepsis. Langenbecks-Arch-Surg. 2006, 391:83-87
28．李建国，彭周全，杜朝晖，等．电针足三里激活胆碱能抗炎通路抗大鼠失血性休克的研究．中国中西医结合急救杂志，2006，13：27-31
29．陆士新．我国对食管癌研究的贡献．首都医药，1998，5：14-15
1．吴光宗，戴桂康．现代科学技术革命与当代社会．北京航空航天大学出版社，2004，4：1-323
2．安维复．从国家创新体系看现代科学技术革命．中国社会科学．2000，5：100-113
3. Wang LD, Bin Yue W, Zhou Y, et al. Endoscopic screening and determination of p53 and proliferating cell nuclear antigen in esophageal multistage carcinogenesis: a comparative study between high- and low-risk populations in Henan, northern China. Dis Esoph, 2002, 15:80-84
4．王立东，郑树．食管癌研究的历史回顾和哲学思考．医学与哲学，2001，22：1-5．
5．杨光华．病理学．人民卫生出版社，2002，1：108-118
6. Zheng Z, Guernsey JR, Riddell DC, et al. Genetic polymorphisms of phase Ⅰ and Ⅱ metabolizing enzymes in adenocarcinomas of the esophgogastric junction. IN: Pinotti H W, eds. Recent advances in diseases of the esophagus. Brazil: Modomond, 2001,117-123
7．孙超，刘宾，王立东．河南食管癌高发区老年与青年食管癌患者P53蛋白和Rb蛋白表达的变化．中华老年医学杂志，2003，22：274-276
8. Nemoto T, Kitagawa M, Hasegawa M, et al. Expression of IAP family in esophageal cancer. Exp Mol Pathol, 2004, 76:253-259
9．李秀香，刘广庆，周刚．食管癌病人精神状态异常与脑电图变化．现代电生理杂志，2000，7：24-25．
10．吕双红，周岩，刘少君．食管鳞癌组织中肽能神经支配研究．解剖学报，2000，33：249-253
11. Christian Otten, Lucia Migliazza, Huimin Xia, et al. Neural crest-derived defects in experimental esophageal atresia. Pediatr Res., 2000, 47: 178.
12. Qi BQ, Merei J, Hasthorpe S, et al. The vagus and recurrent laryngeal nerves in the rodent experimental model of esophageal atresia. J Pedia Surg., 1977, 32: 1580-1586.
13. Fang MZ, Liu C, Song Y, et al. Over-expression of gastrin-releasing peptide in human esophageal squamous cell carcinomas. Carcinogenesis, 2004, 25: 865-871.
14. Ebihara Y, Miyamoto M, Fukunaga A, et al. DARPP-32 expression arises after a phase of dysplasia in oesophageal squamous cell carcinoma. Br J Cancer, 2004, 59:119-123
15. Okumura T, Shmada Y, Imamura M, et al. Neurotrophin receptor p75(NTR) characterizes human esophageal keratinocyte stem cells in vitro. Oncogene, 2003, 22:4017-4026
16．庄则豪，王立东，曹世华，等．食管癌细胞环氧合酶2表达与丝裂霉素C诱导有关．中华内科杂志，2003，42：701-704
References
1． Wang LD,Lipkin M,Qui SL,et al.Labeling index and labeling distribution of cells in esophageal epithelium of individuals at increased risk for esophageal cancer in Huixian,China.Cancer Res,1990,50:2651-2653
2． Wang LD,Qiu SL,Yang GR,et al.A randomized double-blind intervention study on the effect of calcium supplementation on esophageal precancerous lesions in a high-risk population in China.Cancer Epidemiol Biomarkers Prev,1993,2(1) :71-78
3． Wang LD,Shi ST,Zhou Q,et al.Changes in p53 and cyclin D1 protein levels and cell proliferation in different stages of human esophageal and gastric-cardia carcinogenesis.Int J Cancer,1994,59:514-519
4． Wang LD,Zhou Q,Feng CW,et al.Intervention and follow-up on human esophageal precancerous lesions in Henan,northern China,a high-incidence area for esophageal cancer.Gan To Kagaku Ryoho,2002,29:159-172
5． Tao DM.Research evolvement of epidemiology for the relationship between epithelium hyperplasia and carcinogenesis.Zhongliu Fang zhi Magazine,2001． 1:1009-4571
6． Wang LD,Guo HQ,Zhou Q.The lapse of esophageal epithelium hyperplasia,6 years follow-up for 66 cases of epithelium hyperplasia.Journal of Henan medical University,1991,26:328-330
7． Tao MD,Xu YZ,Gu YK,ect.Research for 46161cases of carcinogenesis time and ratio of the esophageal normal and hyperplasia epidermis,zhong liu fang zhi yanjiu,1997,24:155-156．
8． Wang LD.Comprehension and pondering of research for esophageal cancer in Henan.J Zhengzhou Univ(Med sci),2002,37:717-729,2006,41:1-5．
9． Wang LD,Zheng S,Zheng ZY,Casson AG.Primary adenocarcinomas of lower esophagus,esophagogastric junction and gastric cardia:in special reference to China.World J Gastroenterol,2003,9:1156-1164．
10． Wang LD,Zheng F.Mechanism of esophagealand gastric cardia canceration in high-incidence area in Henan.J Zhengzhou Univ(Med sci),2002,37:717-729
11． Wang LD,Hong JY,Qiu SL,et al.Accumulation of p53 protein in human esophageal precancerous lesions:a possible early biomarker for carcinogenesis.Cancer Res.,1993,53:1783-1787
12． Wang ZY,Wang LD,Lee MJ,et al.Inhibition of N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in rats by green and black tea.Carcinogenesis,1995,16:2143-2148
13． Wang DC,Wang LD,Jia YY,et al.Immunohistochemical studies on endocrine-like tumor cells in colorectal carcinomas.Chin J New Gastroenterol,1997,5:30-31
14． Wang LD,Zhou Q,Li J,et al.Preliminary studies of Ras protein expression in esophageal squamous cell carcinoma.Journal of Henan medical University,1997,32:43-45
15． Wang LD,Yang WC,Zhou Q,et al.Alterations of WAF-1 and P53 in human esophageal cancerous and precancerous lesions.Journal of Henan medical University,1997,32:11-14
16． Wang LD,Zhou Q,Hong JY,et al.p53 protein accumulation and gene mutations in multifocal esophageal precancerous lesions from symptom-free subjects in a high incidence area for esophageal carcinoma in Henan,China Cancer,1996,77:1244-1249
17． Fan ZM,Chen P,Liu XL,et al.Alterations of expression of p53,BrdU,P21~(wafl),and PCNA protein in esophageal cancer and precancerous lesions.Journal of Zhengzhou University,2006,41:27-29
18． Wang LD.Studies on mechanism and prevention and cure of esophageal canceration.Chin J New Gastroenterol,1996,4:9-10
19． Blot WJ,Devesa SS,Kneller RW,et al.Rising incidence of adenocarcinoma of the esophagus and gastric cardia.JAMA,1991,265:1287
20． Wang LD,Wang JK,Jiao XY,et al.Incidence of intestinal metaplasia adjacent to gastric cardia adenocarcinoma.J Zhengzhou Uni,2006,41:14-16．
21． Wang LD,Zhou Q,Yang WC.Immunohistochemical studies on P53 tumor suppressor gene esophageal procancinogenesis.Journal of Xinxiang Medical College,1993,10:4-6
22． Gao,HK.Wang,LD.Zhou Q.,et al.P53 tumor suppressor gene mutation in early esophageal precancerous lesions and carcinomas among high risk population in Henan,China.Cancer Res.,1994,54:342-45
23． Wang LD,Zhou Q,Chen YL,et al.Studies on expression of epithelial-specific protein of p40 precancerous and cancerous lesions of esophageal and gastric cardia.Journal of Zhengzhou University(Medical sciences),1995,30:101-103
24． Wang LD,Zhou Q,Xing Y,et al.Comparative studies of relationship between the changes of P53 tumor suppressor gene and cell proliferations in esophageal epithelia among subjects from China and the United State.J Henan med Uni,1995,30:121-123
25． Wang LD,Zhou Q,Wang YH,et al.Comparative studies of expression of glutathione S-transferase in esophageal and gastric cardial epithelium among subjects from China and the United State.Journal of Henan medical University,1995,30:124-127
26． Wang LD,Zhou Q,Wang YH,et al.Studies on expression of procancinogenesis genes EGFR,C-Jun and Ras of esophagus in population in high-incidence area in Henan.J Henan med Uni,1995,30:143-145
27． Wang LD,Zhou Q,Li YX,et al Expression of c-erb B2,c-myc cyclin D1 in human esophageal and gastric cardial precancerous lesions.Journal of Henan medical University,1995,30:146-149
28． Wang LD,Zhou Q,Gao SS,et al Preliminary studies of Ras protein expression in human gastric cardial carcinoma.J Henan med Uni,1995,30:149-152．
29． G.Y.Yang,Z.H.Zhang,J.Liao,et al.Immunohistochemical studies on Waflp21,p16,pRb,and p53 in human esophageal carcinomas and neighboring epithelia from a high-risk area in northern China.Int J Cancer,1997,72:746-751
30． L.D.Wang,W.C.Yang,Q.Zhou,et al.Changes of tumor suppressor gene P53 and WAF-1 P21 and cell proliferation in esophageal carcinogenesis in a high-risk population in northern China.Chin.J New Gastroenterol,1997,3:87-89．
31． Wang LD,Wei JP,Zhou Q,et al.Comparative studies of tumor suppressor gene Maspin expression in gastric antrum adenocarcinoma and chronic gastritis.J Henan med Univ,1997,32:40-42
32． Wang LD,Zhou Q,Yang WC,et al.Expression of tumor suppressor gene Maspin in esophageal squamous cell carcimoma.J Henan med Univ,1997,32:48-49
33． Wang LD,Li XF,Zhou Q,et al.Changes in the expression of tumor suppressor gene P16 and Rb in gastric cardia precancerous lesion.J Henan med Univ,1997,32:55-57
34． Zhou Q,Zhou JX,Chen YL,et al.Alteration of tumor suppressor gene p16 and Rb in gastric cancinogesis.Chin J New Gastroenterol,1997,5:705-706
35． Zhou JX,Chen YL,,Wang ZH,et al.Correlation between alteration of tumor suppressor gene p53,p16 and biological behavior of gastric cancer.Chin J New Gastroenterol,1997,5:775-776
36． Wang LD,Zhou Q,Li J,et al.Changes of P53 and nm23 in primary gastric cardia adenocarcinoma and lymph node metastatic lesions.J Henan med Univ,1997,32:36-40
37． Wang LD,Li J,Li J,et al.Expression of tumor suppresso rgene Rb and P16 in esophageal precancerous lesions.J Henan med Univ,1997,32:30-33
38． Wang LD,Yu GQ,Gao SS,et al.Changes of tumor suppressor gene P16 and P53 in esophageal cancer and its adjacent cancer tissue.J Henan med Univ,1997,32:34-36
39． Wang LD,Zhou Q,Zhang YC,et al.Comparative studies of p53 alterations and cell proliferation in esophageal epithelia among subjects from high and low incidence areas of esophageal cancer.Chin J New Gastroenterol,1997,5:10
40． L.D.Wang,Q.Zhou,J.P.Wei,et al.Apoptosis and its relationship with cell proliferation,p53,Waf1,p21,bcl-2 and c-myc in esophageal carcinogenesis studied with a high-risk population in northern China[J].World J Gastroenterol,1998;4:287-293
41． Wang LD,Yang WC,Zhou Q,et al.,etal.Relationship between the expression of p53,c-myc,bcl-2 and the apoptosis in esophageal carcinogenesis.J Clin Exp Pathol,1998,14:106-108
42． Zhou JX,Chen YL,Wang LD,et al.Expression of Proliferation Cell Nuclear Antigen in Gastric Epithelia with Different Severity of Lesions.Henan Med Res.,1998,7:32-34
43． Li J,Niu ZX,Zhou Q,et al.Correlation of Tumor Suppressor Gene p53 Protein Accumulation in Primary and Lymph Node Metastatic Esophageal and Gastric Cardia Cancer.Henan Med Res.1998,7:3-5
44． Zhou JX,Chen YL,Wang LD,et al.Expression of tumor suppressor gene p16 and Rb in gastric epithelial cancinogesis.Chin J Dig.Endosc.1998,15:153-155．
45． S.T.Shi,G.Y.Yang,L.D.Wang,et al.Role of p53 gene mutations in human esophageal carcinogenesis:results from immunohistochemical and mutation analyses of carcinomas and nearby non-cancerous lesions.Carcinogenesis,1999,20:591-597
46． E.P.Xing,Y.Nie,Y.Song,et al.Mechanisms of inactivation of p14ARF,p15INK4b and p16INK4a genes in human esophageal squamous cell carcinoma.Clin Cancer Res,1999,5:2704-2713
47． Zhou JX,Wang LD,Chen YL,et al.Expression of P16,Cyclin D1 in gastric cancinogesis.Chin J Clin Oncol,1999,26:218-22．
48． Li J,Zhao ZG,Feng CW,et al.Expression of Ras protein in tissue of esophageal squamous cell carcinoma in Linxian of Henan.World J Gastroenterol,1999,7:714
49． Li J,Zhao ZG,Zhou Q,et al.Express ion of ras prote in in human esophageal cancerous and precancerous lesions.Chin J Clin Oncol Reh,1999,6:11-12
50． Li J,Zhou Q,Wang LD,et al.Expression of Tumor Suppressor Gene Maspin in Esophageal and Gastric Cardia Cancer.Cancer research on prevention and treatment,J Zhengzhou Uni(Med Sci),1999,26:1-2
51． Li J,Feng CW,Li SY,et al.Expression of nm23-H_1 protein in human esophageal squamous cell carcinoma in high incidence area of esophageal cancer in China.J Surg Henan,1999,5:9-11
52． Li J,Feng CW,Zhou Q,et al.Study on ras protein in human esophageal squamous cell carcinoma in northern China.Henan Med Res,1999,8:97-99
53． Li J,C.W.Feng,Z.G.Zhao,et al.A preliminary study on ras protein expression in human esophageal cancer and precancerous lesions.World J Gastroenterol 2000,6:278-280
54． LI J,Yang WC,Feng CW,et al.Preliminary study on tumor suppressor gene maspin in esophageal squamous cell carcinomas.Chin J Cancer Prev Treat,2000,7:13-15
55． Q.Zhou,L.D.Wang,F.Du,et al.Changes of TGF-(?)1 and T(?)RII Expression in the Esophageal Precancerous and Cancerous Lesions:a Study of a High-Risk Population in Henan,northern China.Dis Esoph,2002,15:74-79
56． Song ZB,Gao SS,Wang LD,et al.Correlation between p53,PCNA and the prognosis of esophageal carcinoma from the subjects in high-incidence area.Henan Med Res,2002,11:97-100
57． Chen H,Li JL.Guo HQ,et al.Alterations of p53 in concurrent carcinomas of the esophagus and gastric cardia from the same patient in Linzhou,Henan Province,a high-incidence area for esophageal cancer.Henan Med Res,2002,11:7-9
58． An JY,Wang LD,He XW,et al.Expression of PTEN protein in esophageal squamous cell carcinomas and gastric cardia adenocarcinoma tissues from high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:750-752
59． Zhuang ZH,Wang LD,Wang QM,et al.Expression of cyclooxygenase-2 protein in human esophageal carcinoma tissues on the subjects at high2incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:753-755
60． Zhuang ZH,Wang LD,Gao SS,et al.Expression of MUC1 in esophageal and gastric cardiac carcinoma tissues from high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:774-777
61． Feng CW,Qi YJ,Guo RF,et al.Preliminary analysis on phosphotyrosine protein in esophageal cancer and adjacent non-cancer tissues from the patients at high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci), 2002,37:769-771
62． Sun C,Liu B,Wang LD,et al.Expression of P53 and Rb protein in esophageal squamous cell carcinomas from the young and old patient s at high2incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:748-750
63． Wang LD,Liu B,Guo RF,et al.Changes of C2erbB2 and c-myc expression in esophageal and gastriccardia carcinogenesis from the subjects at high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:739-74
64． Qin YR,Liu Z,Guo HQ,,et al.Expression of p27 gene and cyclinE in esophageal precancerous lesions from the subjects at high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:763-765
65． Yue WB,GAO SS,Liu B,et al.Alterations of retinoid acid receptor( RARB)expression in esophageal precancerous and cancerous lesions from the subjects at high and low incidence areas for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:766-769．
66． Zhou Q,BaiYM,Wang LD,et al.Alterations of MUC3 in gastric-cardia precancerous and cancerous lesions:a comparative study between the high-and low-risk populations.J Zhengzhou Uni(Med Sci),2003,38:324-326
67． Zhou Q,Zheng ZY,Wang LD,et al.Prevalence of genetic polymorphisms of CYP1A1 and 2E1 in subjects with gastric cardia-adenocarcinoma at Linzhou,Henan.J Zhengzhou Uni(Med Sci),2003,38:317-320
68． Zhou Q,Zheng ZY,Wang LD,et al.p53 protein accumulation and p53 gene mutation in esophageal and gastric cardia cancer from the patients at Linzhou,Henan.J Zhengzhou Uni(Med Sci),2003,38:313-31．
69． Zhou Q,Bai YM,Wang LD,et al.Alterations of C-erbB2 in gastrocardia precancerous and cancerous lesions:a comparative study between the high-and low-risk populations.J Zhengzhou Uni(Med Sci),2003,38:335-337
70． Sun C,Liu B,Wang LD,et al.Changes of P53 and Rb proteins expression in esophageal squamous cell carcinoma of the elderly and the young patients at high-risk area for the cancer in Henan Province.Chin J Geriatr,2003;22:274-276
71． Li XF,Li JC,Wang LD,et al.Expression of P53,P16 and P21-WAF-1 in cardiac carcinoma tissues.J Zhengzhou Uni(Med Sci),2003,38:761-763
72． Li JX,He X,Wang LD,Qin YR,et al.Expression of cyclin D1 protein and mRNA in cancer and adjacent tissues in the same patient in Linzhou,a high incidence area for esophageal cancer in northern China.Henan Med Res.2003;12:197-200
73． Xie DL,Wang LD,Li JX,et al.Studies of relationship between gastric cardia adenocarcinoma and p15 gene methylation in patients at high-incidence area.Chin J Trad Chin West Med,2004,12:2222-2225
74． Sun C,Wang LD,WangQM,et al.Expression of P53 and Rb protein in esophageal precancerosis and carcinoma tissue of patients and relation to their ages.World J Gastroenter,2004,12:2222-2225
75． Yi XN,Wang LD,Song ZB,et al.Survivin expression in esophageal squamous cell carcinoma of patients from Linzhou city,a high-incidence area for esophageal cancer in northern China.J Zhengzhou Uni(Med Sci),2004;39:953-95．
76． Li JX,Li YJ,Qin YR,et al.FHIT mRNA and protein expression in esophageal carcinoma and matched adjacent normal esophageal tissues at high-incidence area in Henan Province.World Chin J Digestol,2005,13:1417-1420
77． Sun C,Wang LD,WangQM,et al.Expression of RAR(?)and MUC1 protein in esophageal carcinoma tissue in the young and elderly patients.Chin J Dig,2005;25:620-621
78． Lv XD,Wang LD,Qi YJ,et al.Expression of mdm2,bcl-2,bax and p53 protein in the cancer tissues of concurrent carcinomas of the esophagus and gastric cardia.Chin J Cancer Prev Treat,2006,13:5-8
79． Li J,Jiao XY,Du F,et al.Expressive changes of NF-kB protein in the esophageal carcinoma and precancerosis tissue of the subjects at high-incidence area in Henan.J Zhengzhou Uni(Med Sci),2006,41:22-24
80． Wang NB,Gao SS,He X,et al.Expression of Rb in esophagus and gastric cardia concurrent carcinomas tissue from high incidence area of esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2006,41:24-26
81． Chang ZW,Wang LD,Gao SS,et al.Expression of FHIT and p53 in patients with familial esophageal cancer.J Zhengzhou Uni(Med Sci),2006,41:24-26
82． Lv XD,He X,Li JL,et al.Expression ofmdm2,CyclinD1 and p16 in concurrent cancers of esophagus and gastric cardia.J Zhengzhou Uni(Med Sci),2006,41:22-24
83． Fang ZM,Wu DL,Gao SS,et al.Expression of ATP7B in human gastric cardia adenocarcinoma tissue from high incidence area in Henan.J Zhengzhou Uni(Med Sci),2006,41:54-5
84． Qin YR,Li YX,Wang LD,et al.Expression of c-myc,hTERT and c2MET in esophageal squamous cell carcinoma and lymph node metastatic tissue.J Zhengzhou Uni(Med Sci),2006,41:34-36
85． Feng XS,Gao SG,Ma BG,et al.Expression of p53 and PCNA in intestinal metaplasia tissue adjacent to gastric cardia adenocarcinoma and in gastric cardia biopsy tissue.J Zhengzhou Uni(Med Sci),2006,41:30-31
86． Li JL,Li YF,Qin YR,et al.Expression of C-erbB-2 protein and mRNA in esophageal cancer tissue.J Zhengzhou Uni(Med Sci),2006,41:117-119
87． Kawakubo H,Ozawa S,Ando N,et al.Alterations of p53,cyclin D1 and pRB expression in the carcinogenesis of esophageal squamous cell carcinoma.Oncol Rep,2005,14:1453-1459
88． Geddert H,Kiel S,Heep HJ,et al.The role of p63 and deltaNp63(p40) protein expression and gene amplification in esophageal carcinogenesis.Hum Pathol,2003,34:850-856
89． Wang L,Zhang W,Wang W,et al.A study on cyclooxygenase-2 protein expression in esophageal carcinoma and premalignant lesions.Zhonghua Zhong Liu Za Zhi,2001,23:14-16
90． Mori M,Mimori K,Shiraishi T,et al.Altered expression of Fhit in carcinoma and precarcinomatous lesions of the esophagus.Cancer Res,2000,60:1177-1182
91． Shirakawa Y,Naomoto Y,Kimura M,et al.Topological analysis of p21WAF1/CIP1 expression in esophageal squamous dysplasia.Clin Cancer Res,2000,6:541-550
92． zur Hausen A,Sarbia M,Heep H,et al.Retmoblastoma-protein(prb)expression and prognosis in squamous-cell carcinomas of the esophagus.Int J Cancer,1999,84:618-622
93． Mandard AM,Marnay J,Lebeau C,et al.Expression of p53 in oesophageal squamous epithelium from surgical specimens resected for squamous cell carcinoma of the oesophagus,with special reference to uninvolved mucosa.J Pathol,1997,181:153-157
94． Volant A,Nousbaum JB,Giroux MA,et al.p53 protein accumulation in oesophageal squamous cell carcinomas and precancerous lesions.J Clin Pathol,1995,48:531-534
95． Ikeda Y,Kuwano H,Ikebe M,et al.Immunohistochemical detection of CEA,CA19-9,and DF3 in esophageal carcinoma limited to the submucosal layer.J Surg Oncol,1994,56:7-12
96． Burg-Kurland CL,Purnell DM,Combs JW,et al.Immunocytochemical evaluation of human esophageal neoplasms and preneoplastic lesions for beta-chorionic gonadotropin,placental lactogen,alpha-fetoprotein,carcinoembryonic antigen,and nonspecific cross-reacting antigen.Cancer Res,1986,46:2936-2943
97． Matsumoto M,Furihata M,Ohtsuki Y,et al.Immunohistochemical characterization of p57KIP2 expression in human esophageal squamous cell carcinoma.Anticancer Res,2000,20:1947-1952
98． Koide N,Koike S,Adachi W,et al.Immunohistochemical expression of bcl-2 protein in squamous cell carcinoma and basaloid carcinoma of the esophagus.Surg Today,1997;27:685-691
99． Itakura Y,Sasano H,Abe K,et al.Cytokeratin immunolocalization and lectin binding studies in oesophageal squamous dysplasia.Histopathology,1996,29:3-10
100． Itakura Y,Sasano F,Date F,et al.DNA ploidy,P53 expression,and cellular proliferation in normal epithelium and squamous dysplasia of non-cancerous and cancerous human oesophagi.Anticancer Res,1996,16:201-208 101． Jin YL,Zhang W,Liu BQ,et al.Abnormal expression of p53,Ki67 and iNOS in human esophageal carcimoma in situ and pre-malignant lesions.Chin J Oncol,2001,23:129-131 102． Zhang SN,Wang YH,Wang YL,et al.Studies of oncogene and stroma reaction in the esophageal carcinoma and precancerosis.J Weif Med Uni,1999,21:16-18 103． Liu FX,Huang XL,Du XG,et al.A study on expression of PCNA in precancerous lesions of esophageal mucosas.Cancer Res Prev Treat,1998,26:102-104 104． Liu XP,Jiang Y,Zhang XZ,et al.Immunohistochemical study on expression of GST-π in esophageal carcinoma and precancerosis.Chin J Clin Onco Reh,1997,4:11-12 105． Dong YB,Liu SF.Expression of p53 in human esophageal carcinoma and pre-malignant lesions tissue.Chin J Onco,1996,18:58-61 106． Xu F,Li JC,Jin HL,et al.Expression significance of GST-p in esophageal carcinoma and precancerous lesion.Chin J Dig.Endosc.,1995,15:138-139 107． Zhang HY,Li SS,Ren XH,et al.Expressions of Mcm5 and PCNA in the biopsy tissues of esophagus.J Zhengzhou Uni(Med Sci),2004,39:364-366 108． Huang H,Wang LF,Tian HM,et al.Expression of retinoic acid receptor28mRNA and p16,p53,Ki67 proteins in esophageal carcinoma and its precursor lesions.Chin J Oncol,2005,27:152-155 109． Wang LD,Zhou Q,Gou RY,et al.Reproducibility of an esophageal biopsy sampling procedure in a high-incidence area for esophageal cancer in northern China.Cancer Epid Biomarkers Prev.1996,5:405-406 110． Wang LD,Zheng S,Liu B,et al.Analysis on colour endoscopic mass survey and surgically resected esophageal specimen on the subjects at high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:730-732
111． Wang LD,Zheng S.Esophageal cancer research:historical review and its reflect on philosophy.Med Phil,2001,22:1-5 112． Du F,Wang LD,Qi YJ,et al.Testing of the characteristic change of several auto-antibodies in serum from patients suffered from the esophageal and gastric cardia cancer and precancerosis with tumor-related antigen microarray.China J Cancer Prev Treat,2006,(In press)
113． Wang LD,Liu B,Feng CW,et al.Histopathological comparison of the lesions at esophagus,gastric cardiaand antrum on the subjects from Linzhou,Henan and the Linzhou migrated subjects from Changzhi,Shanxi.J Zhengzhou Uni(Med Sci),2006,41:5-9
2．袁媛，张卫东，郗园林．生活事件与食管癌发生的病例对照研究．河南医科大学学报，2001，36：74-76
3．周春锋，夏全，李林海．安县食管癌危险因素的病例对照研究．江苏预防医学，1999，10：14-15
4．俞鲁谊．社会心理与癌症：性格情绪与癌症。中国肿瘤，1992，：27-29
5. Qubain SW, Natsugoe S, Matsumoto S, et al. Micrometastases in the cervical lymph nodes in esophageal squamous cell carcinoma. Dis Esophagus, 2001, 14: 143-148
6. Wang LD, Zhou Q, Feng CW, et al. Intervention and follow-up on human esophageal precancerous lesions in Henan, northern China, a high-incidence area for esophageal cancer. Gan To Kagaku Ryoho, 2002, 29:159-172
7．李秀香，刘广庆，周刚．食管癌病人精神状态异常与脑电图变化．现代电生理学杂志，2000，12：227-228
8. Oliveira RC, Oliveira R, Ferreira CM, et al. Involvement of 5-HT(2) serotonergic receptors of the nucleus raphe magnus and nucleus reticularis gigantocellularis/paragigantocellularis complex neural networks in the antinociceptive phenomenon that follows the post-ictal immobility syndrome. Exp Neurol, 2006, 201:144-153
9. Sampliner RE, Spechler SJ. Controversies in barrett,s esophagus and barret, scancer. World organization for specialized studies on diseases of the esophagus in 8th world congress, 2006, September 3-6
10. Kessler W, Traeger T, Westerholt A, et al. The vagal nerve as a link between the nervous and immune system in the instance of polymicrobial sepsis. Langenbecks Arch Surg, 2006, 391:83-87
11. Guarini S, Altavilla D, Cainazzo MM, et al. Efferent vagal fibre stimulation blunts nuclear factor JB activation and protects against hypovolemic hemorrhagic shock. Circulation, 2003, 107:1189-1194
12．崔忠厚，孙玉鹗，黄孝迈，等．25例电视胸腔镜手术的初步结果．中华外科杂志，1994，32：584-586
13. Balaji NS, Crookes PF, Banki F, et al. A safe and noninvasive test for vagal integrity revisited. Arch Surg, 2002, 137:954-959
14. Yamamoto S, Kawahara K, Maekawa T, et al. Minimally invasive esophagectomy for stage Ⅰ and Ⅱ esophageal cancer. Ann Thorac Surg, 2005, 80: 2070-2075
15．杜贾军，孟龙，陈景寒，等．手辅助电视胸腔镜行食管癌切除术．中华外科杂志，2005，43：351-353
16．王国范，张百江，杨文锋，等．保留迷走神经加胃底重建预防食管贲门癌术后胃食管返流的研究．肿瘤防治杂志，2005，12：218-221
17．蒋跃光．食管疾病．重庆出版社(重庆)，1998：85-86
18. Christian Often, Lucia Migliazza, Huimin Xia, et al. Neural crest-derived defects in experimental esophageal atresia. Pediatr Res, 2000, 47:178-183
19. Qi BQ, Merei J, Hasthorpe S, et al. The vagus and recurrent laryngeal nerves in the rodent experimental model of esophageal atresia. J Pedia Surg, 1977, 32:1580-1586
20．李建国，彭周全，杜朝晖，等．电针足三里激活胆碱能抗炎通路抗大鼠失血性休克的研究．中国中西医结合急救杂志，2006，13：27-31
21. Nordin C, Sjodin I. CSF monoamine patterns in pathological gamblers and healthy controls. J Psychiatr Res. 2006, 40:454-459
22．吕保来，吴爱群，曹莉，等．慢性不可预见性应激对大鼠皮质及海马酪氨酸硝基化的影响解剖学杂志，2006，29：473-475
23. Jeffrey H, Shlley M, Sidney H, et al. Dysfunctional attitudes and 5-HT2A receptors during depression self-harm. Am J Psychiatry, 2003, 160:90-99
24. 24.. Sanchez C, Diaz NJ, Avila J. Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function. Prog Neurobiol, 2000, 61:133-148
25. Richard B, Presland, Richard J. Making sense of the epithelial barrier: what molecular biology and genetics tell us about the functions of oral mucosal and epidermal tissues. Transfer of Advances in Sciences into Dental Education. J Dent Edu, 2002, 66:564-574
26. David JP, Beverly AD, Richard BP. Functional Analysis of the Profilaggrin N-Terminal Peptide: Identification of Domains that Regulate Nuclear and Cytoplasmic Distribution. Journal of Investigative Dermatology, 2002, 119: 661-669
27. Trmbino S, Cesario A, Margaritora S, et al. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesot helioma cells: role of mitogenactivated protein kinase pathway. Cancer Res, 2004, 64:135-145
28. Takita M, Muramatsu I. Alteration of brain nicotine receptor is induced by immobilization stress and nicotinic in rats.Brain Res, 1995, 681:190-192
29. Christian W, Yasutaka H, Dick R. Acetylcholine Increases Intracellular Ca~(2+) Via Nicotinic Receptors in Cultured PDF-Containing Clock Neurons of Drosophila. J Neurophysiol, 2004, 91:912-923
30. Tourovskaia A, Kosar TF, Folch A. Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin. Biophys J, 2006, 90:2192-2198
31．胡昕，吕建新．乙酞胆碱及其拮抗剂对人SK-N-SH细胞增殖及mAchR 1表达的影响．实验生物学报，2005，38：287-296
32. Kinnula VL, Lehtonen S, Sormunen R, et al. Overexpression of peroxiredoxins Ⅰ, Ⅱ, Ⅲ, Ⅴ, and Ⅵ in malignant mesothelioma. J Pathol, 2002, 196:316-323
33. Yanagawa T, lwasa S, lshii T, et al. Peroxiredoxin Ⅰ expression in oral cancer: a potential new tumor marker. Cancer Lett, 2000, 156:27-35
34．沈传陆，朱俊．Prx 1高表达对人PC3前列腺癌细胞氧化损伤的作用．东南大学学报(医学版)，2005，24：377-380
35. Raynal P, Pollard HB. Annexin: The problem of assessing the biological role for gene family of multifunctional calcium and phospholipid-2 binding proteins. Biochem Biophys Acta, 1994, 1197:63-93
36. Cole SP, Pinkoshi MJ, Bhardwaj G, et al. Elevated expression of annexin Ⅱ in a multidrug resistant small cell lung cancer cell line. Br J Cancer, 1992, 65: 498-502
37. Cole SP, Pinkoshi MJ, Bhardwaj G, et al. Elevated expression of annexin Ⅱ in a multidrug resistant small cell lung cancer cell line. Br J Cancer, 1992, 65: 498-502
38．王立东，郑树，刘宾，等．河南食管癌高发区居民食管色素镜检查及手术标本碘染色结果分析．郑州大学学报医学版，2002，37：730-732
39．李质磬，于桂臣．食管神经支配特点的光、电镜观察．解剖学杂志，1996，19：539-542
40．松尾．消化管 endocrine，paracrine，neucrine．医学，1982，120：110
41. Pollard TD, Peters B, Kirfel J, et al. Complete cytolysis and neonatal lethality in keratin 5 knockout mice reveal its fundamental role in skin integrity and in epidermolysis bullosa simplex. Mol Biol Cell, 2001, 12:1775-1789
42. Strnad P, Windoffer R, Leube R E. Induction of rapid and reversible cytokeratin filament network remodeling by inhibition of tyrosine phosphatases. J Cell Sci, 2002, 115:4133-4148
1． Whittaker VP.Handbuch der experimente llenpharma-kologie Bd.Berlin:Springer,1963:1-39
2． Sastry BVR,Sadavongvivad C.Cholinergic systems in non-nervous tissues.Pharmacol Rev,1979,30:65-132
3． Miyazawa A,Fujiyoshi Y,Unwin N.Structure and gating mechanism of the acetylcholine receptor pore.Nature,2003,423:949-955
4． Wessle RI,Kirkpatrick CJ,Racke K.Non-neuronal acetylcholine,a locally acting molecule widely distributed in biological systems:expression and function in human.Pharmacol Ther,1998,77:9279
5． Klapproth H,Remheimer T,Metzen J,et al.Non-neuronal acetylcholine,a signalling molecule synthesized bysurface cell s of rat and man.Naunyn Schmiedebergs Arch Pharmacol,1997,355:515-523
6． Wessle RI,Kilbinger H,Bttinger F,et al.The non-neuronal cholinegic system in humans:expression,function and pathophysiology.Life Sci,2003,72:2055-2061
7． Trmbino S,Cesario A,Margaritora S,et al.Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesot helioma cells:role of mitogen-activated protein kinase pathway.Cancer Res,2004,64:135-145
8． Takita M,Muramatsu I.Alteration of brain nicotine receptor is induced by immobilization stress and nicotinic in rats.Brain Res,1995,681:190-192
9． Christian W,Yasutaka H,Dick RN.Acetylcholine Increases Intracellular Ca~(2+)Via Nicotinic Receptors in Cultured PDF-Containing Clock Neurons of Drosophila.J Neurophysiol,2004,91:912-923
10． Tourovskaia A,Kosar TF,Folch A.Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin.Biophys J,2006,90:2192-2198
11． Engel AG,Ohno K,Sine SM.Congenital myasthenic syndromes:experiments of nature.J Physiol Paris,1998,92:113-117
12．张栩，丛志强，李海峰，等．重症肌无力与胸腺外恶性肿瘤相关性的研究．中华神经科杂志，2005，22：263-265
13. Catherine C, Krishna S, David R, et al. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the β-adrenergic pathway. Circulation. 1998;98:1329-1334
14. Fu Y, Hwang B, Chiu Y, et al. Norepinephrine induces apoptosis in neonatal rat cardiomyocytes through a reactive oxygen species-caspase signaling pathway. Cardiovascular Research, 2004, 62:558-567
15. Dincer HE, Gangopadhyay N, Wang R, et al. Norepinephrine induces alveolar epithelial apoptosis mediated by alpha-, beta-, and angiotensin receptor activation.. Am J Physiol Lung Cell Mol Physiol, 2001, 281:1624-1630
16. Singal T, Dhalla NS, Tappia PS. Norepinephrine-induced changes in gene expression of phospholipase C in cardiomyocytes. J Mol Cell Cardiol, 2006, 41: 126-37
17. Viemari JC, Ramirez JM. Norepinephrine differentially modulates different types of respiratory pacemaker and nonpacemaker neurons. J Neurophysiol, 2006, 95: 2070-82
18．吴爱群，王立东，常志伟，等．乙酰胆碱与去甲肾上腺素对EC109细胞分化的调节作用及其可能性机制．中华肿瘤防治杂志，2007，14：36-39
19．张驰宇，张高红，杨敏，等．四步法消除SYBRGreenl实时定量RT-PCR中引物二聚体的影响．中国生物化学与分子生物学报，2004，20：387-392
20．张蓓．实时荧光定量PCR的研究进展及其应用．国外医学临床生物化学与检验学分册，2003，24：327-329
21．欧阳松应，杨冬，欧阳红生，等．实时荧光定量PCR技术及其应用生命的化学，2004，24：74-76
22. David JP, Beverly AD, Richard BE Functional Analysis of the Profilaggrin N-Terminal Peptide: Identification of Domains that Regulate Nuclear and Cytoplasmic Distribution. Journal of Investigative Dermatology, 2002, 119: 661-669
23. Raynal P, Pollard HB. Annexin: The problem of assessing the biological role for gene family of multifunctional calcium and phospholipid-2 binding proteins. Biochem Biophys Acta, 1994, 1197:63-93
24. Hajjar KA, Krishnan S. Annexin Ⅱ: A mediator of the plasmin/plasminogen activator system. Trends Cardiovasc Med, 1999, 9:128-138
25. Kang HM, Choi KS, Kassam G, et al. Role of annexin Ⅱ tetramer in plasminogen activation. Trends Cardiovasc Med, 1999, 9:92-102
26. Mai JX, Waisman DM, Sloane BF. Cell surface complex of cathepsin B/annexin Ⅱ tetramer in malignant progression. Biochem Biophys Acta 2000, 149:301-322
27. Siever DA, Efickson HP. Extracellular annexin Ⅱ. Int J Biochem Cell Biol, 1997, 29:1219-1223
28. 28. Kinnula VL, Lehtonen S, Sormunen R, et al. Overexpression of peroxiredoxins Ⅰ, Ⅱ, Ⅲ, Ⅴ, and Ⅵ in malignant mesothelioma. J Pathol, 2002, 196:316-323
29．沈传陆，朱俊．Prx 1高表达对人PC3前列腺癌细胞氧化损伤的作用．东南大学学报(医学版)，2005，24：377-380
30. Kumoto K, Sawada H, Yamada Y, et al. Annexin Ⅱ overexpression is correlated with poor prognosis in human gastric carcinoma. Anticancer Res, 2001, 21: 1339-1345
31. Takita M, Muramatsu I. Alteration of brain nicotine receptor is induced by immobilization stress and nicotinic in rats. Brain Res, 1995, 681:190-192
32. David JP, Beverly AD, Richard BP. Functional Analysis of the Profilaggrin N-Terminal Peptide: Identification of Domains that Regulate Nuclear and Cytoplasmic Distribution. J Invest Dermatol, 2002,119:661-669
33. Emoto K, Yamaka Y, Sawada H, et al. Annexin Ⅱ overexpression correlates with stromal tenascin-C overexpression: a prognostic marker in colorectal carcinoma. Cancer, 2001, 92:1419-1426
34. Reeves SA, Chave-Kappel C, Davis R, et al. Developmental regulation of annexin Ⅱ in human brain and expression in high grade glioma. Caner Res, 1992, 52:6871-6876
35. Frohlich M, Motte P, Galvin K, et al. Enhanced expression of the protein-kinase substrate p36 in human hepatocellular carcinoma. Mol Cell Biol, 1990, 10: 3216-3223
36. Cole SP, Pinkoshi MJ, Bhardwaj G, et al. Elevated expression of annexin Ⅱ in a multidrug resistant small cell lung cancer cell line. Br J Cancer, 1992, 65: 498-502
37．李习玲，连小华，张诚，等．Annexin Ⅱ在人表皮角质形成细胞分化中的表达．第四军医大学学报，2004，25：1921-1924
1. 1 H ilger RA, Scheulen M E, Strum berg D. The Ras Raf-M EK-ERK pathway in the treatment of cancer. Onkologie, 2002, 25: 511-518
2. Sherr C J. Principles of tumor suppression. Cell. 2004 Jan 23; 116: 235-46
3．龚小卫，姜勇．丝裂原活化蛋白激酶(MAPK)生物学功能的结构基础．中国生物化学与分子生物学报，2003，19：5-11
4. Duanmu DQ, Li ZH, Wang JZ, et al. MAPK signal transduction pathway: the study progress. J Cell Biol, 2002, 24: 151-154
5. Mueller H, Flury N, Eppenberger CS, et al. Potential prognostic value of mitogen activated protein kinase activity for disease free survival of primary breast cancer patients. Int J Cancer, 2000, 89: 384-388
6．李印 周清华 孙芝琳，等．靶向抑制ERK1／2信号传导通路对人高转移大细胞肺癌细胞株L9981恶性表型的影响．中国肺癌杂志，2005，8：504-509
7. Dincer HE, Gangopadhyay N, Wang R, et al. Norepinephrine (NE) induces apoptosis. Am J Physiol Lung Cell Mol Physiol. 2001, 281: 1624-1630
8. Trmbino S, Cesario A, Margaritora S, et al. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesot helioma cells: role of mitogenactivated protein kinase pathway. Cancer Res, 2004, 64: 135-145
9．吴爱群，王立东，常志伟，等．乙酰胆碱与去甲肾上腺素对食管癌细胞分化的调制作用及其机制．中华肿瘤防治杂志，2007，14：36-39
10. Acqua ML, Smith KE, Gorski JA, et, al. Home EA, Regulation of neuronal PKA signaling through AKAP targeting dynamics. Eur J Cell Biol. 2006, 85: 627-33
11. Pei J J, Braak H, An WL, et al. Up-regulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degeneration in Alzheimer's disease. Brain Res Mol Brain Res. 2002, 30; 109: 45-55
12. Trmbino S, Cesario A, Margadtora S, et al. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesot helioma cells: role of mitogenactivated protein kinase pathway. Cancer Res, 2004, 64: 135-145
13. 13. Wessle RI, Kirkpatrick CJ, Racke K. Non-neuronal acetylcholine, a locally acting molecule widely distributed in biological systems: expression and function in human. Pharmacol Ther, 1998, 77: 923-919
14. Klapproth H, Reinheimer T, Metzen J, et al. Non-neuronal acetylcholine, a signalling molecule synthesized by surface cells of rat and man. Naunyn Schmiedebergs Arch Pharmacol, 1997, 355:515-523
15. 15. Takita M, Muramatsu I. Alteration of brain nicotine receptor is induced by immobilization stress and nicotinic in rats. Brain Res, 1995, 681:1902192.
16. Knobloch O, Tischner R. Characterization of nitrate reductase deficient mutants of Chlorella Sorokini. Physiol,1989, 89:78 6-791
17. 16. Zhang XJ, Yang L, Caen JP, et al. Induction of acelcholinestease expression during apoptosis in various cell types. Cell Dezth Differ, 2002, 9:790～800
18．胡昕，吕建新．乙酞胆碱及其拮抗剂对人SK-N-SH细胞增殖及mAchR 1表达的影响．实验生物学报，2005，38：287～296
19. DeFea K A, Zalevsky J, Thoma MS, et al. β-Arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol, 2000, 148,: 1267-1282
20. Girnita L, Shenoy S K, Sehat B, et al. beta-Arrestin and Mdm2 Mediate IGF-1 Receptor- stimulated ERK Activation and cell cycle progression. J. Biol. Chem. 2007, 282(1): 11329-11338
21. Arora, P., Ricks, T. K., Trejo, J. Protease-activated receptor signalling, endocytic sorting and dysregulation in cancer. J. Cell Sci. 2007, 120:921-928
22. Goetzl EJ. Diverse pathways for nuclear signaling by G protein-coupled receptors and their ligands. FASEB J. 2007, 21:638-642
23. Ma, L., Pei, G. beta-arrestin signaling and regulation of transcription. J. Cell Sci.. 2007 80:1214-1221
24. Kinnula VL, Lehtonen S, Sormunen R, et al. Overexpression of peroxiredoxins Ⅰ, Ⅱ, Ⅲ, Ⅴ, and Ⅵ in malignant mesothelioma. J Pathol, 2002, 196:316-323
25．沈传陆，朱俊．Prx 1高表达对人PC3前列腺癌细胞氧化损伤的作用．东南大学学报(医学版)，2005，24：377-380
26. 26. Richard B, Presland, Richard J. Making sense of the epithelial barrier: what molecular biology and genetics tell us about the functions of oral mucosal and epidermal tissues. J Dent Edu, 2002, 66:564-574
1．林东昕．中国食管癌分子流行病学研究．中华流行病学杂志，2003，24：939-943
2．王立东，郑树．食管癌研究的历史回顾和哲学思考．医学与哲学，2001，22：1-5
3．袁媛，张卫东，郗园林．生活事件与食管癌发生的病例对照研究．河南医科大学学报，2001，36：74-76
4. Wang LD, Zhou Q, Feng CW, et al. Intervention and follow-up on human esophageal precancerous lesions in Henan, northern China, a high-incidence area for esophageal cancer. Gan To Kagaku Ryoho, 2002, 29:159-172
5．李秀香，刘广庆，周刚．食管癌病人精神状态异常与脑电图变化．现代电生理杂志，2000，7：24-25
6. Bonne O, Brandes D, Gilboa A, et al. Longitudinal MRI study of hippocapal volume in trauma survivors with depression. Am J Psychiatry, 2001, 158: 1248-1251
7. Nordin C, Sjodin I. CSF monoamine patterns in pathological gamblers and healthy controls. J-Psychiatr-Res. 2006, 40:454-459
8. Jeffrey H, Shlley M, Sidney H, et al. Dysfunctional attitudes and 5-HT2A receptors during depression self-harm. Am J psychiatry, 2003, 160:90-99
9. Keeney A, Jessop D-S, Harbuz M-S, et al. Differential effects of acute and chronic social defeat stress on hypothalamic-pituitary-adrenal axis function and hippocampal serotonin release in mice. J-Neuroendocrinol. 2006, 18:330-338
10. Ferretti C, Blengio M, Gamalero SR, et al. Biochemical and behaviour changes induced by acute stress in chronic stress model of depression: the effect of amitriptyline. Eur J phanacol. 1995, 280:19-26
11. Sarbaka SN, Saha K. Moderate exercise and chronic stress produce counteractive effects on different areas of the brain by acting through various neurotransmitter receptor subtypes: a hypothesis. Theor-Biol-Med-Model. 2006, 3:33-38
12．吕保来，吴爱群，曹莉，等．慢性不可预见性应激对大鼠皮质及海马酪氨酸硝基化的影响．解剖学杂志，2006，29：473-475
13. Weisstaub NV, Zhou M, Lira A, et al. Cortical 5-HT2A receptor signaling modulates anxiety-like behaviors in mice. Science. 2006, 313:536-540
14. Gilbertson MW, Shenton MW, Ciszeuski A, et al. Smaller hippocampal volume predicts pathologic vulnerability to psychological trauma. Nat Neurosci, 2002, 5: 1242-1247
15．李鹂，涂自良，杜士明，等．氟西汀对实验性抑郁症大鼠抑郁行为及海马神经元再生的影响．医药导报，2006，25：280-282
16. Marcoli M, Cervetto C, Paluzzi P, et al. Nitric oxide-evoked glutamate release and cGMP production in cerebellar slices: control by presynaptic 5-HT1D receptors. Neurochem-Int, 2006, 49:12-19
17. Munoz Castaneda JR, Montilla P, Padillo FJ, et al. Role of serotonin in cerebral oxidative stress in rats. Acta-Neurobiol-Exp-(Wars). 2006, 66:1-6
18. de-Oliveira RC, de-Oliveira R, Ferreira CM, et al. Involvement of 5-HT(2) serotonergic receptors of the nucleus raphe magnus and nucleus reticularis gigantocellulads/ paragigantocellularis complex neural networks in the anti-nociceptive phenomenon that follows the post-ictal immobility syndrome. Exp-Neurol. 2006, 201:144-153
19. Monti JM, Jantos H. Effects of activation and blockade of 5-HT2A/2C receptors in the dorsal raphe nucleus on sleep and waking in the rat. Prog-Neuropsychopharmacol-Biol-Psychiatry. 2006, 30:1189-1195
20. Trmbino S, Cesario A, Margaritora S, et al. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesot helioma cells: role of mitogen-activated protein kinase pathway. Cancer Res, 2004, 64:135-145
21．汤燕平，工建国．乙酞胆碱对人胃粘膜上皮细胞和胃腺癌细胞N受体p亚单位的影响．中国应用生理学杂志，2005，21：457-460
22. Takita M, Muramatsu I. Alteration of brain nicotine receptor sinduced by immobilization stress and nicotinic in rats. BrainRes, 1995, 681:190-192
23. Guarini S, Altavilla D, Cainazzo MM, et al. Efferent vagal fibre stimulation blunts nuclear factor JB activation and protects against hypovolemic hemorrhagic shock. Circulation, 2003, 107:1189-1194
24．余炜伟，王立东，齐义军，等．食管鳞癌组织中p16基因缺失及甲基化检测．郑州大学学报(医学版)，2006，41：282-284
25．魏亚宁，舒青，张素珍．亚硝胺类化合物诱导小鼠食管病变与基因组甲基化的关系研究．科学技术与工程，2006，6：2036-2040
26．张栩，丛志强，李海峰，等．重症肌无力与胸腺外恶性肿瘤相关性的研究．中华神经科杂志，2005，22：263-265
27. Kessler W, Traeger T, Westerholt, A, et al. The vagal nerve as a link between the nervous and immune system in the instance of polymicrobial sepsis. Langenbecks-Arch-Surg. 2006, 391:83-87
28．李建国，彭周全，杜朝晖，等．电针足三里激活胆碱能抗炎通路抗大鼠失血性休克的研究．中国中西医结合急救杂志，2006，13：27-31
29．陆士新．我国对食管癌研究的贡献．首都医药，1998，5：14-15
1．吴光宗，戴桂康．现代科学技术革命与当代社会．北京航空航天大学出版社，2004，4：1-323
2．安维复．从国家创新体系看现代科学技术革命．中国社会科学．2000，5：100-113
3. Wang LD, Bin Yue W, Zhou Y, et al. Endoscopic screening and determination of p53 and proliferating cell nuclear antigen in esophageal multistage carcinogenesis: a comparative study between high- and low-risk populations in Henan, northern China. Dis Esoph, 2002, 15:80-84
4．王立东，郑树．食管癌研究的历史回顾和哲学思考．医学与哲学，2001，22：1-5．
5．杨光华．病理学．人民卫生出版社，2002，1：108-118
6. Zheng Z, Guernsey JR, Riddell DC, et al. Genetic polymorphisms of phase Ⅰ and Ⅱ metabolizing enzymes in adenocarcinomas of the esophgogastric junction. IN: Pinotti H W, eds. Recent advances in diseases of the esophagus. Brazil: Modomond, 2001,117-123
7．孙超，刘宾，王立东．河南食管癌高发区老年与青年食管癌患者P53蛋白和Rb蛋白表达的变化．中华老年医学杂志，2003，22：274-276
8. Nemoto T, Kitagawa M, Hasegawa M, et al. Expression of IAP family in esophageal cancer. Exp Mol Pathol, 2004, 76:253-259
9．李秀香，刘广庆，周刚．食管癌病人精神状态异常与脑电图变化．现代电生理杂志，2000，7：24-25．
10．吕双红，周岩，刘少君．食管鳞癌组织中肽能神经支配研究．解剖学报，2000，33：249-253
11. Christian Otten, Lucia Migliazza, Huimin Xia, et al. Neural crest-derived defects in experimental esophageal atresia. Pediatr Res., 2000, 47: 178.
12. Qi BQ, Merei J, Hasthorpe S, et al. The vagus and recurrent laryngeal nerves in the rodent experimental model of esophageal atresia. J Pedia Surg., 1977, 32: 1580-1586.
13. Fang MZ, Liu C, Song Y, et al. Over-expression of gastrin-releasing peptide in human esophageal squamous cell carcinomas. Carcinogenesis, 2004, 25: 865-871.
14. Ebihara Y, Miyamoto M, Fukunaga A, et al. DARPP-32 expression arises after a phase of dysplasia in oesophageal squamous cell carcinoma. Br J Cancer, 2004, 59:119-123
15. Okumura T, Shmada Y, Imamura M, et al. Neurotrophin receptor p75(NTR) characterizes human esophageal keratinocyte stem cells in vitro. Oncogene, 2003, 22:4017-4026
16．庄则豪，王立东，曹世华，等．食管癌细胞环氧合酶2表达与丝裂霉素C诱导有关．中华内科杂志，2003，42：701-704
References
1． Wang LD,Lipkin M,Qui SL,et al.Labeling index and labeling distribution of cells in esophageal epithelium of individuals at increased risk for esophageal cancer in Huixian,China.Cancer Res,1990,50:2651-2653
2． Wang LD,Qiu SL,Yang GR,et al.A randomized double-blind intervention study on the effect of calcium supplementation on esophageal precancerous lesions in a high-risk population in China.Cancer Epidemiol Biomarkers Prev,1993,2(1) :71-78
3． Wang LD,Shi ST,Zhou Q,et al.Changes in p53 and cyclin D1 protein levels and cell proliferation in different stages of human esophageal and gastric-cardia carcinogenesis.Int J Cancer,1994,59:514-519
4． Wang LD,Zhou Q,Feng CW,et al.Intervention and follow-up on human esophageal precancerous lesions in Henan,northern China,a high-incidence area for esophageal cancer.Gan To Kagaku Ryoho,2002,29:159-172
5． Tao DM.Research evolvement of epidemiology for the relationship between epithelium hyperplasia and carcinogenesis.Zhongliu Fang zhi Magazine,2001． 1:1009-4571
6． Wang LD,Guo HQ,Zhou Q.The lapse of esophageal epithelium hyperplasia,6 years follow-up for 66 cases of epithelium hyperplasia.Journal of Henan medical University,1991,26:328-330
7． Tao MD,Xu YZ,Gu YK,ect.Research for 46161cases of carcinogenesis time and ratio of the esophageal normal and hyperplasia epidermis,zhong liu fang zhi yanjiu,1997,24:155-156．
8． Wang LD.Comprehension and pondering of research for esophageal cancer in Henan.J Zhengzhou Univ(Med sci),2002,37:717-729,2006,41:1-5．
9． Wang LD,Zheng S,Zheng ZY,Casson AG.Primary adenocarcinomas of lower esophagus,esophagogastric junction and gastric cardia:in special reference to China.World J Gastroenterol,2003,9:1156-1164．
10． Wang LD,Zheng F.Mechanism of esophagealand gastric cardia canceration in high-incidence area in Henan.J Zhengzhou Univ(Med sci),2002,37:717-729
11． Wang LD,Hong JY,Qiu SL,et al.Accumulation of p53 protein in human esophageal precancerous lesions:a possible early biomarker for carcinogenesis.Cancer Res.,1993,53:1783-1787
12． Wang ZY,Wang LD,Lee MJ,et al.Inhibition of N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in rats by green and black tea.Carcinogenesis,1995,16:2143-2148
13． Wang DC,Wang LD,Jia YY,et al.Immunohistochemical studies on endocrine-like tumor cells in colorectal carcinomas.Chin J New Gastroenterol,1997,5:30-31
14． Wang LD,Zhou Q,Li J,et al.Preliminary studies of Ras protein expression in esophageal squamous cell carcinoma.Journal of Henan medical University,1997,32:43-45
15． Wang LD,Yang WC,Zhou Q,et al.Alterations of WAF-1 and P53 in human esophageal cancerous and precancerous lesions.Journal of Henan medical University,1997,32:11-14
16． Wang LD,Zhou Q,Hong JY,et al.p53 protein accumulation and gene mutations in multifocal esophageal precancerous lesions from symptom-free subjects in a high incidence area for esophageal carcinoma in Henan,China Cancer,1996,77:1244-1249
17． Fan ZM,Chen P,Liu XL,et al.Alterations of expression of p53,BrdU,P21~(wafl),and PCNA protein in esophageal cancer and precancerous lesions.Journal of Zhengzhou University,2006,41:27-29
18． Wang LD.Studies on mechanism and prevention and cure of esophageal canceration.Chin J New Gastroenterol,1996,4:9-10
19． Blot WJ,Devesa SS,Kneller RW,et al.Rising incidence of adenocarcinoma of the esophagus and gastric cardia.JAMA,1991,265:1287
20． Wang LD,Wang JK,Jiao XY,et al.Incidence of intestinal metaplasia adjacent to gastric cardia adenocarcinoma.J Zhengzhou Uni,2006,41:14-16．
21． Wang LD,Zhou Q,Yang WC.Immunohistochemical studies on P53 tumor suppressor gene esophageal procancinogenesis.Journal of Xinxiang Medical College,1993,10:4-6
22． Gao,HK.Wang,LD.Zhou Q.,et al.P53 tumor suppressor gene mutation in early esophageal precancerous lesions and carcinomas among high risk population in Henan,China.Cancer Res.,1994,54:342-45
23． Wang LD,Zhou Q,Chen YL,et al.Studies on expression of epithelial-specific protein of p40 precancerous and cancerous lesions of esophageal and gastric cardia.Journal of Zhengzhou University(Medical sciences),1995,30:101-103
24． Wang LD,Zhou Q,Xing Y,et al.Comparative studies of relationship between the changes of P53 tumor suppressor gene and cell proliferations in esophageal epithelia among subjects from China and the United State.J Henan med Uni,1995,30:121-123
25． Wang LD,Zhou Q,Wang YH,et al.Comparative studies of expression of glutathione S-transferase in esophageal and gastric cardial epithelium among subjects from China and the United State.Journal of Henan medical University,1995,30:124-127
26． Wang LD,Zhou Q,Wang YH,et al.Studies on expression of procancinogenesis genes EGFR,C-Jun and Ras of esophagus in population in high-incidence area in Henan.J Henan med Uni,1995,30:143-145
27． Wang LD,Zhou Q,Li YX,et al Expression of c-erb B2,c-myc cyclin D1 in human esophageal and gastric cardial precancerous lesions.Journal of Henan medical University,1995,30:146-149
28． Wang LD,Zhou Q,Gao SS,et al Preliminary studies of Ras protein expression in human gastric cardial carcinoma.J Henan med Uni,1995,30:149-152．
29． G.Y.Yang,Z.H.Zhang,J.Liao,et al.Immunohistochemical studies on Waflp21,p16,pRb,and p53 in human esophageal carcinomas and neighboring epithelia from a high-risk area in northern China.Int J Cancer,1997,72:746-751
30． L.D.Wang,W.C.Yang,Q.Zhou,et al.Changes of tumor suppressor gene P53 and WAF-1 P21 and cell proliferation in esophageal carcinogenesis in a high-risk population in northern China.Chin.J New Gastroenterol,1997,3:87-89．
31． Wang LD,Wei JP,Zhou Q,et al.Comparative studies of tumor suppressor gene Maspin expression in gastric antrum adenocarcinoma and chronic gastritis.J Henan med Univ,1997,32:40-42
32． Wang LD,Zhou Q,Yang WC,et al.Expression of tumor suppressor gene Maspin in esophageal squamous cell carcimoma.J Henan med Univ,1997,32:48-49
33． Wang LD,Li XF,Zhou Q,et al.Changes in the expression of tumor suppressor gene P16 and Rb in gastric cardia precancerous lesion.J Henan med Univ,1997,32:55-57
34． Zhou Q,Zhou JX,Chen YL,et al.Alteration of tumor suppressor gene p16 and Rb in gastric cancinogesis.Chin J New Gastroenterol,1997,5:705-706
35． Zhou JX,Chen YL,,Wang ZH,et al.Correlation between alteration of tumor suppressor gene p53,p16 and biological behavior of gastric cancer.Chin J New Gastroenterol,1997,5:775-776
36． Wang LD,Zhou Q,Li J,et al.Changes of P53 and nm23 in primary gastric cardia adenocarcinoma and lymph node metastatic lesions.J Henan med Univ,1997,32:36-40
37． Wang LD,Li J,Li J,et al.Expression of tumor suppresso rgene Rb and P16 in esophageal precancerous lesions.J Henan med Univ,1997,32:30-33
38． Wang LD,Yu GQ,Gao SS,et al.Changes of tumor suppressor gene P16 and P53 in esophageal cancer and its adjacent cancer tissue.J Henan med Univ,1997,32:34-36
39． Wang LD,Zhou Q,Zhang YC,et al.Comparative studies of p53 alterations and cell proliferation in esophageal epithelia among subjects from high and low incidence areas of esophageal cancer.Chin J New Gastroenterol,1997,5:10
40． L.D.Wang,Q.Zhou,J.P.Wei,et al.Apoptosis and its relationship with cell proliferation,p53,Waf1,p21,bcl-2 and c-myc in esophageal carcinogenesis studied with a high-risk population in northern China[J].World J Gastroenterol,1998;4:287-293
41． Wang LD,Yang WC,Zhou Q,et al.,etal.Relationship between the expression of p53,c-myc,bcl-2 and the apoptosis in esophageal carcinogenesis.J Clin Exp Pathol,1998,14:106-108
42． Zhou JX,Chen YL,Wang LD,et al.Expression of Proliferation Cell Nuclear Antigen in Gastric Epithelia with Different Severity of Lesions.Henan Med Res.,1998,7:32-34
43． Li J,Niu ZX,Zhou Q,et al.Correlation of Tumor Suppressor Gene p53 Protein Accumulation in Primary and Lymph Node Metastatic Esophageal and Gastric Cardia Cancer.Henan Med Res.1998,7:3-5
44． Zhou JX,Chen YL,Wang LD,et al.Expression of tumor suppressor gene p16 and Rb in gastric epithelial cancinogesis.Chin J Dig.Endosc.1998,15:153-155．
45． S.T.Shi,G.Y.Yang,L.D.Wang,et al.Role of p53 gene mutations in human esophageal carcinogenesis:results from immunohistochemical and mutation analyses of carcinomas and nearby non-cancerous lesions.Carcinogenesis,1999,20:591-597
46． E.P.Xing,Y.Nie,Y.Song,et al.Mechanisms of inactivation of p14ARF,p15INK4b and p16INK4a genes in human esophageal squamous cell carcinoma.Clin Cancer Res,1999,5:2704-2713
47． Zhou JX,Wang LD,Chen YL,et al.Expression of P16,Cyclin D1 in gastric cancinogesis.Chin J Clin Oncol,1999,26:218-22．
48． Li J,Zhao ZG,Feng CW,et al.Expression of Ras protein in tissue of esophageal squamous cell carcinoma in Linxian of Henan.World J Gastroenterol,1999,7:714
49． Li J,Zhao ZG,Zhou Q,et al.Express ion of ras prote in in human esophageal cancerous and precancerous lesions.Chin J Clin Oncol Reh,1999,6:11-12
50． Li J,Zhou Q,Wang LD,et al.Expression of Tumor Suppressor Gene Maspin in Esophageal and Gastric Cardia Cancer.Cancer research on prevention and treatment,J Zhengzhou Uni(Med Sci),1999,26:1-2
51． Li J,Feng CW,Li SY,et al.Expression of nm23-H_1 protein in human esophageal squamous cell carcinoma in high incidence area of esophageal cancer in China.J Surg Henan,1999,5:9-11
52． Li J,Feng CW,Zhou Q,et al.Study on ras protein in human esophageal squamous cell carcinoma in northern China.Henan Med Res,1999,8:97-99
53． Li J,C.W.Feng,Z.G.Zhao,et al.A preliminary study on ras protein expression in human esophageal cancer and precancerous lesions.World J Gastroenterol 2000,6:278-280
54． LI J,Yang WC,Feng CW,et al.Preliminary study on tumor suppressor gene maspin in esophageal squamous cell carcinomas.Chin J Cancer Prev Treat,2000,7:13-15
55． Q.Zhou,L.D.Wang,F.Du,et al.Changes of TGF-(?)1 and T(?)RII Expression in the Esophageal Precancerous and Cancerous Lesions:a Study of a High-Risk Population in Henan,northern China.Dis Esoph,2002,15:74-79
56． Song ZB,Gao SS,Wang LD,et al.Correlation between p53,PCNA and the prognosis of esophageal carcinoma from the subjects in high-incidence area.Henan Med Res,2002,11:97-100
57． Chen H,Li JL.Guo HQ,et al.Alterations of p53 in concurrent carcinomas of the esophagus and gastric cardia from the same patient in Linzhou,Henan Province,a high-incidence area for esophageal cancer.Henan Med Res,2002,11:7-9
58． An JY,Wang LD,He XW,et al.Expression of PTEN protein in esophageal squamous cell carcinomas and gastric cardia adenocarcinoma tissues from high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:750-752
59． Zhuang ZH,Wang LD,Wang QM,et al.Expression of cyclooxygenase-2 protein in human esophageal carcinoma tissues on the subjects at high2incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:753-755
60． Zhuang ZH,Wang LD,Gao SS,et al.Expression of MUC1 in esophageal and gastric cardiac carcinoma tissues from high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:774-777
61． Feng CW,Qi YJ,Guo RF,et al.Preliminary analysis on phosphotyrosine protein in esophageal cancer and adjacent non-cancer tissues from the patients at high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci), 2002,37:769-771
62． Sun C,Liu B,Wang LD,et al.Expression of P53 and Rb protein in esophageal squamous cell carcinomas from the young and old patient s at high2incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:748-750
63． Wang LD,Liu B,Guo RF,et al.Changes of C2erbB2 and c-myc expression in esophageal and gastriccardia carcinogenesis from the subjects at high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:739-74
64． Qin YR,Liu Z,Guo HQ,,et al.Expression of p27 gene and cyclinE in esophageal precancerous lesions from the subjects at high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:763-765
65． Yue WB,GAO SS,Liu B,et al.Alterations of retinoid acid receptor( RARB)expression in esophageal precancerous and cancerous lesions from the subjects at high and low incidence areas for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:766-769．
66． Zhou Q,BaiYM,Wang LD,et al.Alterations of MUC3 in gastric-cardia precancerous and cancerous lesions:a comparative study between the high-and low-risk populations.J Zhengzhou Uni(Med Sci),2003,38:324-326
67． Zhou Q,Zheng ZY,Wang LD,et al.Prevalence of genetic polymorphisms of CYP1A1 and 2E1 in subjects with gastric cardia-adenocarcinoma at Linzhou,Henan.J Zhengzhou Uni(Med Sci),2003,38:317-320
68． Zhou Q,Zheng ZY,Wang LD,et al.p53 protein accumulation and p53 gene mutation in esophageal and gastric cardia cancer from the patients at Linzhou,Henan.J Zhengzhou Uni(Med Sci),2003,38:313-31．
69． Zhou Q,Bai YM,Wang LD,et al.Alterations of C-erbB2 in gastrocardia precancerous and cancerous lesions:a comparative study between the high-and low-risk populations.J Zhengzhou Uni(Med Sci),2003,38:335-337
70． Sun C,Liu B,Wang LD,et al.Changes of P53 and Rb proteins expression in esophageal squamous cell carcinoma of the elderly and the young patients at high-risk area for the cancer in Henan Province.Chin J Geriatr,2003;22:274-276
71． Li XF,Li JC,Wang LD,et al.Expression of P53,P16 and P21-WAF-1 in cardiac carcinoma tissues.J Zhengzhou Uni(Med Sci),2003,38:761-763
72． Li JX,He X,Wang LD,Qin YR,et al.Expression of cyclin D1 protein and mRNA in cancer and adjacent tissues in the same patient in Linzhou,a high incidence area for esophageal cancer in northern China.Henan Med Res.2003;12:197-200
73． Xie DL,Wang LD,Li JX,et al.Studies of relationship between gastric cardia adenocarcinoma and p15 gene methylation in patients at high-incidence area.Chin J Trad Chin West Med,2004,12:2222-2225
74． Sun C,Wang LD,WangQM,et al.Expression of P53 and Rb protein in esophageal precancerosis and carcinoma tissue of patients and relation to their ages.World J Gastroenter,2004,12:2222-2225
75． Yi XN,Wang LD,Song ZB,et al.Survivin expression in esophageal squamous cell carcinoma of patients from Linzhou city,a high-incidence area for esophageal cancer in northern China.J Zhengzhou Uni(Med Sci),2004;39:953-95．
76． Li JX,Li YJ,Qin YR,et al.FHIT mRNA and protein expression in esophageal carcinoma and matched adjacent normal esophageal tissues at high-incidence area in Henan Province.World Chin J Digestol,2005,13:1417-1420
77． Sun C,Wang LD,WangQM,et al.Expression of RAR(?)and MUC1 protein in esophageal carcinoma tissue in the young and elderly patients.Chin J Dig,2005;25:620-621
78． Lv XD,Wang LD,Qi YJ,et al.Expression of mdm2,bcl-2,bax and p53 protein in the cancer tissues of concurrent carcinomas of the esophagus and gastric cardia.Chin J Cancer Prev Treat,2006,13:5-8
79． Li J,Jiao XY,Du F,et al.Expressive changes of NF-kB protein in the esophageal carcinoma and precancerosis tissue of the subjects at high-incidence area in Henan.J Zhengzhou Uni(Med Sci),2006,41:22-24
80． Wang NB,Gao SS,He X,et al.Expression of Rb in esophagus and gastric cardia concurrent carcinomas tissue from high incidence area of esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2006,41:24-26
81． Chang ZW,Wang LD,Gao SS,et al.Expression of FHIT and p53 in patients with familial esophageal cancer.J Zhengzhou Uni(Med Sci),2006,41:24-26
82． Lv XD,He X,Li JL,et al.Expression ofmdm2,CyclinD1 and p16 in concurrent cancers of esophagus and gastric cardia.J Zhengzhou Uni(Med Sci),2006,41:22-24
83． Fang ZM,Wu DL,Gao SS,et al.Expression of ATP7B in human gastric cardia adenocarcinoma tissue from high incidence area in Henan.J Zhengzhou Uni(Med Sci),2006,41:54-5
84． Qin YR,Li YX,Wang LD,et al.Expression of c-myc,hTERT and c2MET in esophageal squamous cell carcinoma and lymph node metastatic tissue.J Zhengzhou Uni(Med Sci),2006,41:34-36
85． Feng XS,Gao SG,Ma BG,et al.Expression of p53 and PCNA in intestinal metaplasia tissue adjacent to gastric cardia adenocarcinoma and in gastric cardia biopsy tissue.J Zhengzhou Uni(Med Sci),2006,41:30-31
86． Li JL,Li YF,Qin YR,et al.Expression of C-erbB-2 protein and mRNA in esophageal cancer tissue.J Zhengzhou Uni(Med Sci),2006,41:117-119
87． Kawakubo H,Ozawa S,Ando N,et al.Alterations of p53,cyclin D1 and pRB expression in the carcinogenesis of esophageal squamous cell carcinoma.Oncol Rep,2005,14:1453-1459
88． Geddert H,Kiel S,Heep HJ,et al.The role of p63 and deltaNp63(p40) protein expression and gene amplification in esophageal carcinogenesis.Hum Pathol,2003,34:850-856
89． Wang L,Zhang W,Wang W,et al.A study on cyclooxygenase-2 protein expression in esophageal carcinoma and premalignant lesions.Zhonghua Zhong Liu Za Zhi,2001,23:14-16
90． Mori M,Mimori K,Shiraishi T,et al.Altered expression of Fhit in carcinoma and precarcinomatous lesions of the esophagus.Cancer Res,2000,60:1177-1182
91． Shirakawa Y,Naomoto Y,Kimura M,et al.Topological analysis of p21WAF1/CIP1 expression in esophageal squamous dysplasia.Clin Cancer Res,2000,6:541-550
92． zur Hausen A,Sarbia M,Heep H,et al.Retmoblastoma-protein(prb)expression and prognosis in squamous-cell carcinomas of the esophagus.Int J Cancer,1999,84:618-622
93． Mandard AM,Marnay J,Lebeau C,et al.Expression of p53 in oesophageal squamous epithelium from surgical specimens resected for squamous cell carcinoma of the oesophagus,with special reference to uninvolved mucosa.J Pathol,1997,181:153-157
94． Volant A,Nousbaum JB,Giroux MA,et al.p53 protein accumulation in oesophageal squamous cell carcinomas and precancerous lesions.J Clin Pathol,1995,48:531-534
95． Ikeda Y,Kuwano H,Ikebe M,et al.Immunohistochemical detection of CEA,CA19-9,and DF3 in esophageal carcinoma limited to the submucosal layer.J Surg Oncol,1994,56:7-12
96． Burg-Kurland CL,Purnell DM,Combs JW,et al.Immunocytochemical evaluation of human esophageal neoplasms and preneoplastic lesions for beta-chorionic gonadotropin,placental lactogen,alpha-fetoprotein,carcinoembryonic antigen,and nonspecific cross-reacting antigen.Cancer Res,1986,46:2936-2943
97． Matsumoto M,Furihata M,Ohtsuki Y,et al.Immunohistochemical characterization of p57KIP2 expression in human esophageal squamous cell carcinoma.Anticancer Res,2000,20:1947-1952
98． Koide N,Koike S,Adachi W,et al.Immunohistochemical expression of bcl-2 protein in squamous cell carcinoma and basaloid carcinoma of the esophagus.Surg Today,1997;27:685-691
99． Itakura Y,Sasano H,Abe K,et al.Cytokeratin immunolocalization and lectin binding studies in oesophageal squamous dysplasia.Histopathology,1996,29:3-10
100． Itakura Y,Sasano F,Date F,et al.DNA ploidy,P53 expression,and cellular proliferation in normal epithelium and squamous dysplasia of non-cancerous and cancerous human oesophagi.Anticancer Res,1996,16:201-208 101． Jin YL,Zhang W,Liu BQ,et al.Abnormal expression of p53,Ki67 and iNOS in human esophageal carcimoma in situ and pre-malignant lesions.Chin J Oncol,2001,23:129-131 102． Zhang SN,Wang YH,Wang YL,et al.Studies of oncogene and stroma reaction in the esophageal carcinoma and precancerosis.J Weif Med Uni,1999,21:16-18 103． Liu FX,Huang XL,Du XG,et al.A study on expression of PCNA in precancerous lesions of esophageal mucosas.Cancer Res Prev Treat,1998,26:102-104 104． Liu XP,Jiang Y,Zhang XZ,et al.Immunohistochemical study on expression of GST-π in esophageal carcinoma and precancerosis.Chin J Clin Onco Reh,1997,4:11-12 105． Dong YB,Liu SF.Expression of p53 in human esophageal carcinoma and pre-malignant lesions tissue.Chin J Onco,1996,18:58-61 106． Xu F,Li JC,Jin HL,et al.Expression significance of GST-p in esophageal carcinoma and precancerous lesion.Chin J Dig.Endosc.,1995,15:138-139 107． Zhang HY,Li SS,Ren XH,et al.Expressions of Mcm5 and PCNA in the biopsy tissues of esophagus.J Zhengzhou Uni(Med Sci),2004,39:364-366 108． Huang H,Wang LF,Tian HM,et al.Expression of retinoic acid receptor28mRNA and p16,p53,Ki67 proteins in esophageal carcinoma and its precursor lesions.Chin J Oncol,2005,27:152-155 109． Wang LD,Zhou Q,Gou RY,et al.Reproducibility of an esophageal biopsy sampling procedure in a high-incidence area for esophageal cancer in northern China.Cancer Epid Biomarkers Prev.1996,5:405-406 110． Wang LD,Zheng S,Liu B,et al.Analysis on colour endoscopic mass survey and surgically resected esophageal specimen on the subjects at high-incidence area for esophageal cancer in Henan.J Zhengzhou Uni(Med Sci),2002,37:730-732
111． Wang LD,Zheng S.Esophageal cancer research:historical review and its reflect on philosophy.Med Phil,2001,22:1-5 112． Du F,Wang LD,Qi YJ,et al.Testing of the characteristic change of several auto-antibodies in serum from patients suffered from the esophageal and gastric cardia cancer and precancerosis with tumor-related antigen microarray.China J Cancer Prev Treat,2006,(In press)
113． Wang LD,Liu B,Feng CW,et al.Histopathological comparison of the lesions at esophagus,gastric cardiaand antrum on the subjects from Linzhou,Henan and the Linzhou migrated subjects from Changzhi,Shanxi.J Zhengzhou Uni(Med Sci),2006,41:5-9