小檗碱抗人高转移性肺癌细胞诱导的人脐静脉血管内皮细胞凋亡的作用研究
摘要
肿瘤转移是临床抗肿瘤治疗失败和患者死亡的主要原因,因此,控制转移是决定癌症患者预后的关键因素。肿瘤转移是一个复杂的、多步骤的过程。血行转移是肿瘤转移最重要的途径。肿瘤细胞与血管内皮细胞问的相互作用是血行转移最复杂、最重要的环节。已有研究表明,肿瘤细胞转移过程中伴随着血管内皮细胞的皱缩和凋亡,且血管内皮细胞内第二信使Ca~(2+)浓度及pH值的改变与细胞凋亡密切相关。
小檗碱(Berberine)是消化系统感染常用药。近来研究发现,小檗碱还具有抑制肿瘤细胞生长的作用。我们也对其抗肿瘤作用进行了初步研究,发现小檗碱具有抑制肺癌细胞增殖,抑制实体瘤生长及减少肺内转移灶的作用。但其抗肿瘤转移的机制还有待进一步深入研究。
激光扫描共聚焦显微镜(Laser Scanning Confocal Microscope,LSCM)是现代生物学中用于无损伤观察活细胞和组织的一种重要方法。与特异性荧光探针结合,广泛应用于在活细胞层次监测一些重要生化成分的空间分布和随时间的变化,从而获得生命活动过程的信息。本实验将利用共聚焦显微镜研究小檗碱抗人高转移性肺癌细胞(PG细胞)诱导的人脐静脉血管内皮细胞(HUVEC)凋亡的作用,阐明其抗肿瘤转移的可能机制。
1目的
研究小檗碱抗PG细胞上清液诱导的HUVEC凋亡的作用,阐明小檗碱抑制肿瘤细胞转移的机理。利用共聚焦荧光成像技术观察小檗碱对肿瘤转移的影响,从细胞、亚细胞水平上阐明药物与细胞的作用机制,为中药研究开辟一条新的研究途径。
2方法
首先用MTT法筛选出有效影响HUVEC活性的PG细胞培养上清液浓度与作用时间,建立PG细胞上清液诱导HUVEC凋亡模型。用共聚焦荧光成像技术观察小檗碱抗HUVEC凋亡效应,具体方法:药物防治组用不同浓度小檗碱处理HUVEC后,再与PG细胞培养上清液作用。经MTT法检测不同药物浓度、不同药物作用时间下HUVEC的活性,以确定小檗碱防治的最佳药物剂量和最佳作用时间。应用激光扫描共聚焦显微镜技术测定HUVEC凋亡百分率、细胞核形态、细胞内Ca~(2+)浓度及pH值变化。
3结果
(1)小檗碱能够明显降低PG细胞上清液诱导的HUVEC凋亡百分率。
(2)小檗碱可有效减轻HUVEC细胞核染色质浓缩等凋亡形态变化。
(3)小檗碱可缓解PG细胞培养上清液引起的HUVEC内Ca~(2+)浓度升高。
(4)小檗碱可改善PG细胞培养上清液引起的HUVEC内pH值下降。
4结论
小檗碱能够抗PG细胞诱导的HUVEC凋亡,使细胞结构和功能趋于恢复正常,并呈现剂量依赖关系。其作用机制可能是通过调控细胞内Ca~(2+)浓度,改善pH值,从而抑制内皮细胞凋亡,防止肿瘤转移。
Tumor metastasis is the primary cause of failures in antineoplaston and death of patients;therefore,how to control tumor metastasis is the key to decide the prognosis of cancer patients.The tumor metastasis is a complex and multi-step process.Hematogenous metastasis is the most important way.The interaction of tumor cell and the vascular endothelial cell is the most complex and the most important link in hematogenous metastasis.Some research indicate that tumor metastasis is followed with vascular endothelial cell's shrinkage and apoptosis, the second messenger Ca~(2+)density and the pH change in the vascular endothelial cell have a close correlation with apoptosis.
The berberine is a common medication in the digestive system's infection.Some researches indicate that berberine also has the function to restrain the cancer cell's growth in resent years.We have also conducted preliminary study in this function,discovering that the berberine has the function to restrain the lung cancer cell's multiplication,the growth of solid tumor and to reduce lung-metastasis. But its mechanism in anti-tumor metastasis also needs further research.
The Laser Scanning Confocal Microscope(LSCM)is a important method in the modern biology used in the non-damage observation of living cell and organization.Combined with specificity fluorescent probe,it widely applies in monitoring some important biochemical ingredient in spatial distribution and change along with the time in the living cell level,thus to obtain the information of the vital movement process.This experiment will use LSCM to research the protective function that berberine on the PG cell to cause the apoptosis of human umbilical vein endothelial cell(HUVEC),expounds the mechanism to resist tumor metastasis.
1 Goal
To research the protective function which berberine to PG cell causes HUVEC apoptosis,expounded the mechanism that berberine resist tumor metastasis.Using LSCM to observe influence that berberine on the tumor metastasis,to expound the mechanism of action between drug and cell,and open a new research way for the traditional Chinese medicine research.
2 Method
First we use the MTT method to screen the most appropriate density and the time which function between the HUVEC and PG cell to culture supernatant,with the PG cell to culture supernatant and function with the HUVEC,establishing the tumor attacking model.The medicine protection group is using the berberine to process HUVEC before the PG cell culture supernatant function with the HUVEC.We use the MTT method to detect the cell's survival percentage in different medicine density and different medicine response time,to determine that the best medicine dosage and the best response time with the berberine.Then we carries on the observation and analysis aiming directly at the HUVEC percentage of apoptosis,the shape of nucleolus,the Ca2+ density and the pH value change and so.
3 Result
(1)Berberine can obviously reduce the apoptosis percentage of HUVEC attacked by tumor.
(2)Berberine can relieve the deformation of nucleolus,the concentration of chromatin and so on.
(3)Berberine can relieve the calcium overload of HUVEC.
(4)Berberine can restrain the depression of pH value.
4 Conclusion
The berberine can obviously reduce the HUVEC damage caused by tumor,making the cell structure and the function restore normally.One of these kinds of possibly protective mechanisms is to restrain apoptosis through reducing the excessive load of the Ca~(2+)in the cell and the pH reducing.
小檗碱(Berberine)是消化系统感染常用药。近来研究发现,小檗碱还具有抑制肿瘤细胞生长的作用。我们也对其抗肿瘤作用进行了初步研究,发现小檗碱具有抑制肺癌细胞增殖,抑制实体瘤生长及减少肺内转移灶的作用。但其抗肿瘤转移的机制还有待进一步深入研究。
激光扫描共聚焦显微镜(Laser Scanning Confocal Microscope,LSCM)是现代生物学中用于无损伤观察活细胞和组织的一种重要方法。与特异性荧光探针结合,广泛应用于在活细胞层次监测一些重要生化成分的空间分布和随时间的变化,从而获得生命活动过程的信息。本实验将利用共聚焦显微镜研究小檗碱抗人高转移性肺癌细胞(PG细胞)诱导的人脐静脉血管内皮细胞(HUVEC)凋亡的作用,阐明其抗肿瘤转移的可能机制。
1目的
研究小檗碱抗PG细胞上清液诱导的HUVEC凋亡的作用,阐明小檗碱抑制肿瘤细胞转移的机理。利用共聚焦荧光成像技术观察小檗碱对肿瘤转移的影响,从细胞、亚细胞水平上阐明药物与细胞的作用机制,为中药研究开辟一条新的研究途径。
2方法
首先用MTT法筛选出有效影响HUVEC活性的PG细胞培养上清液浓度与作用时间,建立PG细胞上清液诱导HUVEC凋亡模型。用共聚焦荧光成像技术观察小檗碱抗HUVEC凋亡效应,具体方法:药物防治组用不同浓度小檗碱处理HUVEC后,再与PG细胞培养上清液作用。经MTT法检测不同药物浓度、不同药物作用时间下HUVEC的活性,以确定小檗碱防治的最佳药物剂量和最佳作用时间。应用激光扫描共聚焦显微镜技术测定HUVEC凋亡百分率、细胞核形态、细胞内Ca~(2+)浓度及pH值变化。
3结果
(1)小檗碱能够明显降低PG细胞上清液诱导的HUVEC凋亡百分率。
(2)小檗碱可有效减轻HUVEC细胞核染色质浓缩等凋亡形态变化。
(3)小檗碱可缓解PG细胞培养上清液引起的HUVEC内Ca~(2+)浓度升高。
(4)小檗碱可改善PG细胞培养上清液引起的HUVEC内pH值下降。
4结论
小檗碱能够抗PG细胞诱导的HUVEC凋亡,使细胞结构和功能趋于恢复正常,并呈现剂量依赖关系。其作用机制可能是通过调控细胞内Ca~(2+)浓度,改善pH值,从而抑制内皮细胞凋亡,防止肿瘤转移。
Tumor metastasis is the primary cause of failures in antineoplaston and death of patients;therefore,how to control tumor metastasis is the key to decide the prognosis of cancer patients.The tumor metastasis is a complex and multi-step process.Hematogenous metastasis is the most important way.The interaction of tumor cell and the vascular endothelial cell is the most complex and the most important link in hematogenous metastasis.Some research indicate that tumor metastasis is followed with vascular endothelial cell's shrinkage and apoptosis, the second messenger Ca~(2+)density and the pH change in the vascular endothelial cell have a close correlation with apoptosis.
The berberine is a common medication in the digestive system's infection.Some researches indicate that berberine also has the function to restrain the cancer cell's growth in resent years.We have also conducted preliminary study in this function,discovering that the berberine has the function to restrain the lung cancer cell's multiplication,the growth of solid tumor and to reduce lung-metastasis. But its mechanism in anti-tumor metastasis also needs further research.
The Laser Scanning Confocal Microscope(LSCM)is a important method in the modern biology used in the non-damage observation of living cell and organization.Combined with specificity fluorescent probe,it widely applies in monitoring some important biochemical ingredient in spatial distribution and change along with the time in the living cell level,thus to obtain the information of the vital movement process.This experiment will use LSCM to research the protective function that berberine on the PG cell to cause the apoptosis of human umbilical vein endothelial cell(HUVEC),expounds the mechanism to resist tumor metastasis.
1 Goal
To research the protective function which berberine to PG cell causes HUVEC apoptosis,expounded the mechanism that berberine resist tumor metastasis.Using LSCM to observe influence that berberine on the tumor metastasis,to expound the mechanism of action between drug and cell,and open a new research way for the traditional Chinese medicine research.
2 Method
First we use the MTT method to screen the most appropriate density and the time which function between the HUVEC and PG cell to culture supernatant,with the PG cell to culture supernatant and function with the HUVEC,establishing the tumor attacking model.The medicine protection group is using the berberine to process HUVEC before the PG cell culture supernatant function with the HUVEC.We use the MTT method to detect the cell's survival percentage in different medicine density and different medicine response time,to determine that the best medicine dosage and the best response time with the berberine.Then we carries on the observation and analysis aiming directly at the HUVEC percentage of apoptosis,the shape of nucleolus,the Ca2+ density and the pH value change and so.
3 Result
(1)Berberine can obviously reduce the apoptosis percentage of HUVEC attacked by tumor.
(2)Berberine can relieve the deformation of nucleolus,the concentration of chromatin and so on.
(3)Berberine can relieve the calcium overload of HUVEC.
(4)Berberine can restrain the depression of pH value.
4 Conclusion
The berberine can obviously reduce the HUVEC damage caused by tumor,making the cell structure and the function restore normally.One of these kinds of possibly protective mechanisms is to restrain apoptosis through reducing the excessive load of the Ca~(2+)in the cell and the pH reducing.
引文
[1]黄启福.病理学.北京科学出版社,2004:179
[2]王寅,谭侃,程明和,等.中药防治肿瘤转移的研究进展.中草药,2002,33(10):附3-5
[3]辛海,马琴.防治恶性肿瘤转移的理论探讨.中国中医基础医学杂志,2004,10(2):45-47
[4]王文萍,王垂杰,姜良铎,等.肿瘤转移的“痰毒流注”理论形成基础及实践意义.中国中医基础医学杂志,2002,8(5):4-6
[5]王旺胜,李小伟,张卫华.活血化瘀法合收敛固摄法防治肿瘤转移浅探.国医 论坛,2004,19(2):16-17
[6]沈朝萍,刘锦霞,马兰花,等.浅谈活血化瘀中药抗肿瘤转移的思路与应用.国医论坛,2001,16(4):12
[7]陈雨凤,张少华.活血化瘀药抗肿瘤及肿瘤转移的作用机理探讨.陕西中医学院学报,2006,29(2):62-63
[8]林洪生,李树奇,裴迎霞,等.川芎嗪、苦参碱对癌细胞与内皮细胞黏附及黏附因子表达的影响.中国新药杂志,1999,8(6):384
[9]陈文君,欧阳学农.从肿瘤转移基本过程与机制谈活血化瘀法在肿瘤治疗中的应用.中国中医基础医学杂志,2006,12(5):325-326
[10]巨大维,魏品康.清热解毒中药在恶性肿瘤防治中的药用机理与应用.吉林中医药,2007,27(1):60-63
[11]扬新中.中医药防治肿瘤转移的思路.湖北中医药杂志,2002,24(8):8
[12]郑广娟,刘福利,张丹,等.燥湿药抗肿瘤侵袭和转移作用的实验研究.医学研究通讯,2002,31(3):61-63
[13]王江东,郑广娟.燥湿化痰法治疗肿瘤机制初探.山东中医杂志,2002,21(7):393-394
[14]李忠.临床中医肿瘤学.辽宁科学技术出版社,2002:288-293
[15]王志学.中医药抗肿瘤转移治疗的策略.国医论坛,2005,20(1):17-19
[16]刘复兴,王旺胜,胡志萍.固涩药在防治肿瘤转移中的理论初探.江西中医药,2004,35(260):12-13
[17]孙静.收涩中药对小鼠肝癌血道转移的影响.山东中医药大学学报,2007,31(2):162-164
[18]苏成业,刘金友,许洪霞,等.莪术醇在正常大鼠及肿瘤小鼠体内的代谢研究.药学学报,1980,15(5):257
[19]李佩文.中药预防肿瘤转移的临床及基础研究.中国中医药信息杂志,1996,3(12):27-29
[20]陈婷梅,祝彼得.抗白血病中药及天然药物的研究现状.中国中西医结合杂志,1995,15(5):317-320
[21]冯利,林洪生.榄香烯对Lewis肺癌小鼠基底膜及细胞外间质影响的实验研究.中国肿瘤临床,2005,32(15):891-894
[22]刘炳亚,林言箴.苦马豆素抑制胃癌生长及转移的实验研究.中华肿瘤杂志,1998,20(3):169-170
[23]刘红岩,韩锐.金荞麦提取物抑制肿瘤细胞侵袭、转移和HT-1080细胞产生Ⅳ型胶原酶的研究.中国药理学通报,1998.14(1):36-39
[24]王长秀,马润娣,于立坚.土贝母皂苷对人高转移巨细胞肺癌PGCL3细胞侵袭行为的影响.中国临床药理学与治疗学,2006,11(1):40-45
[25]郝钰,徐泊文,吴珺,等.小檗碱对高转移性人巨细胞肺癌PG细胞黏附特性的影响.中国免疫学杂志,2006,22(11):1025-1028
[26]孙婧璟,周信达,刘银坤.丹参对肝癌转移复发防治作用的研究.中国中西医结合杂志,1999.19(5):292-295
[27]林洪生,李树奇,裴迎霞,等.川芎嗪、苦参碱对癌细胞与内皮细胞黏附及黏附分子表达的影响.中国新药杂志,1999,8(6):384-386
[28]周龙恩,刘红岩,徐少锋,等.紫杉醇对MDA—MB-435人乳腺癌高转移细胞粘附,侵袭及迁移能力的影响.中国药理学与毒理学杂志,2001,15(1):51-55
[29]陈清勇,王彦刈,周建英,等.茶多酚对高转移性肺癌细胞与内皮细胞粘附及粘附分子表达的影响.中国微循环,2000,7(1):17-19
[30]叶炯贤,叶红军,林益平,等.刺五加叶皂甙对肝癌癌基因表达的调节作用.中华实验外科杂志,2000,17(5):426-427
[31]毛海婷,张玲,王芸,等.淫羊藿甙和济南假单胞菌制剂调控PG细胞转移相关基因的表达.肿瘤,2000,20(1):13-15
[32]曲迅,郑广娟,刘德山,等.体外罗勒多糖抗人高转移肺癌细胞侵袭转移作用及机制探讨.中国病理生理杂志,2005,21(7):1345-1348
[33]韩建勋,吴军正,李峰,等.8-甲氧基补骨脂素对MEC-1细胞癌基因/抑癌基因表达的影响.实用口腔医学杂志,1999,15(6):457-458
[34]茅家惠,周爱玲,朱玉娟.华蟾素对小鼠H22肝癌移植瘤作用的研究.中国交通医学杂志,2004,18(5):483-485
[35]刘福君,茹祥斌.地黄及六味地黄汤(丸)的免疫药理及抗肿瘤作用.中草药,1996,27(2):116-118
[36]张淑兰,孙云汉,刘晓平,等.冬虫夏草及人工虫草菌丝抗Lewis肺癌的研究.中药通报,1987,12(2):205
[37]董竞成,董晓辉.黄芪注射液与白细胞介素2增强树突细胞抗肿瘤转移作用的比较研究.中国中西医结合杂志,2005,25(3):236-239
[38]Mitani N,Murakanfi K,Yamaura T,et al.Inhibitory effect of berberine on the mediastinal lymph node metastasis produced by orthotopic implantation of Lewis lung carcinoma.Cancer Lett,2001,165(1):35-42
[39]孔庆志,黄涛,黄冬生,等.苦参素对小鼠移植性S180肉瘤血管形成的抑制作用.中国药师,2003,6(12):769-771
[40]冯刚,孔庆志,黄冬生,等.薏苡仁注射液对小鼠移植性S180肉瘤血管形成的抑制作用.肿瘤防治研究,2004,31(4):229-230
[41]薛东波.人参皂苷Rg3抗肿瘤转移作用的研究进展.中国中西医结合外科杂志,2001,7(4):289-290
[42]陈明伟,倪磊.人参皂苷Rg3对肿瘤血管生长调控因子蛋白表达抑制作用的研究.中国中药杂志,2005,30(5):357-360
[43]沃兴德,丁志山.姜黄素及其衍生物抑制肿瘤作用的实验研究.浙江中医学院学报,2005,29(2):53-58
[44]冯敢生,李欣.中药白及提取物抑制肿瘤血管生成机制的实验研究.中华医学杂志,2003,83(5):412-416
[45]丁怡,赵庆亮,李勇刚,等.雷公藤甲素对血管内皮细胞迁移活性的作用.四川大学学报(医学版),2005,36(3):347-350
[46]张前.羟基红花黄色素A抑制新生血管形成的机制研究.北京中医药大学学报,2004,27(3):25-29
[47]江蔚新,蒋燕,季字彬,等.补中益气汤对EAC小鼠癌细胞LPO含量的影响.黑龙江医药,1997,10(2):88-90
[48]林洪生,李树奇,朴炳奎,等.三参冲剂对肺癌转移中内皮细胞及黏附因子的影响.中国肿瘤,1999,8(12):574-576
[49]王彦刈,吴爱姣,许楠,等.复方丹参对高转移性人肺癌细胞与血管内皮细胞黏附及黏附分子表达的影响.中国现代应用药学杂志,2003,5(20):343-345
[50]许建华,范忠泽,孙钰等.肠胃清抗结肠癌转移的机制研究.中国中医药科技,2006,13(3):148-150
[51]许继平,裘维焰,应栩华,等.扶肺煎对原发性肺癌抗转移的临床研究.中国中医药信息杂志,2000(8):40-41
[52]王希胜,张英.山仙颗粒对小鼠黑色素瘤肺转移中nm23/NDPK水平的影响.陕西中医,2006,27(2):244-245
[53]叶向荣,张丹,赵飞.加味血府逐瘀汤抗癌细胞转移的实验研究.山东中医药大学学报,2000,24(6):464-465
[54]陈达理,张绪慧.鳖甲煎丸抗肿瘤血管生成的实验研究.浙江中医杂志,2004,12(12):535-537
[55]徐振晔,王中奇.肺岩宁对晚期非小细胞肺癌生长转移和血清VEGF的影响.上海中医药大学学报,2003,17(3):18-22
[56]王笑民,王征.固本消瘤胶囊抑制小鼠lewis肺癌生长及抗血管生成研究.中国实验方剂学杂志,2004,10(5):50-52
[57]施京红,刘德传.清香散对湿热型肝癌模型大鼠细胞凋亡、肿瘤血管生成的影响.陕西中医,2004,25(1):84-86
[58]刘玉琴,高进,李健生.金龙胶囊抗肿瘤复发、转移的实验研究.北京医学.2005,27(9):554-557
[1]De Vita VT,Hellman S,Rosenberg S.Principles of Oncology(5th Edition).Philadelphia:Lippincott Raven Publisher,1997:135
[2]Gwag BJ,Canzoniero LM,Sensi SL,et al.Calciumion ophores can induce either apoptosis or necrosis in cultured cortical neurons.Neuro science,1999,90(4):1339-1348
[3]曹泽毅.激素受体及临床应用.北京:北京医科大学、中国协和医科大学联合出版社,1993:48-70
[4]Rroemer G,Petit P,Zamzam N,et al.The biochemistry of programmed cell death.FASEB J,1995,9:1277
[5]Gottlieb RA,Nordberg J,Skowronski E,et al.Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.Proc Natl Acad Sci USA,1996,93:654
[6]Barry MA,Reynolds JE,and Eastman A.Etoposide induced apoptosis in human HL 260 cells is associated with intracellular acidification.Cancer Res,1993,53:2349
[7]Gottlieb RA,Giesing H,Zhu J,et al.The acid deoxyribonuclease of neut rophils a possible participant in apoptosis associated genome destruction.Blood,1995,86:2414
[8]Melino G,Annicchiarico Petruzzelli M,Piredda L,et al.Tissue transglutaminase and apoptosis:Senseand antisense transfection studies with human neuro blastoma cells.Mol Cell Biol,1994,14:6584
[9]Paddock SW.Principle and practices of laser scanning confocal microscopy.Mol Biotechnol,2000,16(2):127-149
[10]吴明耀,吴贤英,郑瑞明,等.激光扫描共聚焦显微镜观察免疫组化染色标本.临床与实验病理学杂志,2001,17(6):533-534
[11]司徒镇强,吴军正.细胞培养.西安:世界图书出版西安公司,2007:200
[12]王珊,周春喜,李宁.检测早期细胞凋亡的流式细胞术.现代科学仪器,2003,3:48-50.
[13]赵婷秀,姜霞,王秀莲,等.激光共聚焦显微镜观察FITC-Annexin V/PI双染色法检测人肺癌细胞凋亡.湖北中医学院学报,2005,7(4):9-11
[14]刁志宏,张明霞,朱幼芙.乙型肝炎病毒P22e蛋白对人肝细胞癌细胞株HepG2凋亡的影响.南方医科大学学报,2007,27(11):1649-1652
[15]袁兰.激光扫描共聚焦显微镜技术教程.北京:北京大学医学出版社,2004:79.
[16]王敏,Vogel I,Kalthoff H.尿激酶型纤溶酶原激活因子及抑制剂表达与人大肠癌细胞转移能力的关系.实用肿瘤杂志,2002,17(2):112-114
[17]余琛,洪有采,张慧,等.一种高灵敏度的小檗碱高效液相色谱分析方法.色谱,1994,12(1):37-38
[18]金镇,裴凌,王德智.肿瘤坏死因子对脐静脉血管内皮细胞增殖和细胞内游离钙浓度的影响.中国妇产科杂志,2000,35:657-659
[19]Kroemer G,Petit P,Zamzami N,et al.The biochemistry of programmed cell Death.FASEB J,1995,9(13):1277
[20]Zhu WH,Loh TT.Effects of Na~+/H~+ antiport and intracellular pH in the regulation of HL- 60 cell apoptosis.Biochem Biophys Acta,1995,1269(2):122
[21]KaiserN,Edelman IS.Calicium depedence of glucocorticoid-induced Lymphocytolysis.Proc Natl Acad Sci USA,1977,74:6382642
[22]Squier M KT,Miller ACK,Malkinson AM.Calpain activation in apoptosis.J Cell Physiol,1994,159:2292237
[23]Barry MA,Eastman A.Identification of deoxyribonuclease as an endonuclease involved in apoptosis.Arch Biochem Biophys,1993,300(1):440
[24]Matsubara K,Kubota M,Adachi S,et al.Different mode of cell death by calcium ionophore in human leukemia cell lines:possible role of constitutive endomuclease.Exp Cell Res,1994,210(1):19
[2]王寅,谭侃,程明和,等.中药防治肿瘤转移的研究进展.中草药,2002,33(10):附3-5
[3]辛海,马琴.防治恶性肿瘤转移的理论探讨.中国中医基础医学杂志,2004,10(2):45-47
[4]王文萍,王垂杰,姜良铎,等.肿瘤转移的“痰毒流注”理论形成基础及实践意义.中国中医基础医学杂志,2002,8(5):4-6
[5]王旺胜,李小伟,张卫华.活血化瘀法合收敛固摄法防治肿瘤转移浅探.国医 论坛,2004,19(2):16-17
[6]沈朝萍,刘锦霞,马兰花,等.浅谈活血化瘀中药抗肿瘤转移的思路与应用.国医论坛,2001,16(4):12
[7]陈雨凤,张少华.活血化瘀药抗肿瘤及肿瘤转移的作用机理探讨.陕西中医学院学报,2006,29(2):62-63
[8]林洪生,李树奇,裴迎霞,等.川芎嗪、苦参碱对癌细胞与内皮细胞黏附及黏附因子表达的影响.中国新药杂志,1999,8(6):384
[9]陈文君,欧阳学农.从肿瘤转移基本过程与机制谈活血化瘀法在肿瘤治疗中的应用.中国中医基础医学杂志,2006,12(5):325-326
[10]巨大维,魏品康.清热解毒中药在恶性肿瘤防治中的药用机理与应用.吉林中医药,2007,27(1):60-63
[11]扬新中.中医药防治肿瘤转移的思路.湖北中医药杂志,2002,24(8):8
[12]郑广娟,刘福利,张丹,等.燥湿药抗肿瘤侵袭和转移作用的实验研究.医学研究通讯,2002,31(3):61-63
[13]王江东,郑广娟.燥湿化痰法治疗肿瘤机制初探.山东中医杂志,2002,21(7):393-394
[14]李忠.临床中医肿瘤学.辽宁科学技术出版社,2002:288-293
[15]王志学.中医药抗肿瘤转移治疗的策略.国医论坛,2005,20(1):17-19
[16]刘复兴,王旺胜,胡志萍.固涩药在防治肿瘤转移中的理论初探.江西中医药,2004,35(260):12-13
[17]孙静.收涩中药对小鼠肝癌血道转移的影响.山东中医药大学学报,2007,31(2):162-164
[18]苏成业,刘金友,许洪霞,等.莪术醇在正常大鼠及肿瘤小鼠体内的代谢研究.药学学报,1980,15(5):257
[19]李佩文.中药预防肿瘤转移的临床及基础研究.中国中医药信息杂志,1996,3(12):27-29
[20]陈婷梅,祝彼得.抗白血病中药及天然药物的研究现状.中国中西医结合杂志,1995,15(5):317-320
[21]冯利,林洪生.榄香烯对Lewis肺癌小鼠基底膜及细胞外间质影响的实验研究.中国肿瘤临床,2005,32(15):891-894
[22]刘炳亚,林言箴.苦马豆素抑制胃癌生长及转移的实验研究.中华肿瘤杂志,1998,20(3):169-170
[23]刘红岩,韩锐.金荞麦提取物抑制肿瘤细胞侵袭、转移和HT-1080细胞产生Ⅳ型胶原酶的研究.中国药理学通报,1998.14(1):36-39
[24]王长秀,马润娣,于立坚.土贝母皂苷对人高转移巨细胞肺癌PGCL3细胞侵袭行为的影响.中国临床药理学与治疗学,2006,11(1):40-45
[25]郝钰,徐泊文,吴珺,等.小檗碱对高转移性人巨细胞肺癌PG细胞黏附特性的影响.中国免疫学杂志,2006,22(11):1025-1028
[26]孙婧璟,周信达,刘银坤.丹参对肝癌转移复发防治作用的研究.中国中西医结合杂志,1999.19(5):292-295
[27]林洪生,李树奇,裴迎霞,等.川芎嗪、苦参碱对癌细胞与内皮细胞黏附及黏附分子表达的影响.中国新药杂志,1999,8(6):384-386
[28]周龙恩,刘红岩,徐少锋,等.紫杉醇对MDA—MB-435人乳腺癌高转移细胞粘附,侵袭及迁移能力的影响.中国药理学与毒理学杂志,2001,15(1):51-55
[29]陈清勇,王彦刈,周建英,等.茶多酚对高转移性肺癌细胞与内皮细胞粘附及粘附分子表达的影响.中国微循环,2000,7(1):17-19
[30]叶炯贤,叶红军,林益平,等.刺五加叶皂甙对肝癌癌基因表达的调节作用.中华实验外科杂志,2000,17(5):426-427
[31]毛海婷,张玲,王芸,等.淫羊藿甙和济南假单胞菌制剂调控PG细胞转移相关基因的表达.肿瘤,2000,20(1):13-15
[32]曲迅,郑广娟,刘德山,等.体外罗勒多糖抗人高转移肺癌细胞侵袭转移作用及机制探讨.中国病理生理杂志,2005,21(7):1345-1348
[33]韩建勋,吴军正,李峰,等.8-甲氧基补骨脂素对MEC-1细胞癌基因/抑癌基因表达的影响.实用口腔医学杂志,1999,15(6):457-458
[34]茅家惠,周爱玲,朱玉娟.华蟾素对小鼠H22肝癌移植瘤作用的研究.中国交通医学杂志,2004,18(5):483-485
[35]刘福君,茹祥斌.地黄及六味地黄汤(丸)的免疫药理及抗肿瘤作用.中草药,1996,27(2):116-118
[36]张淑兰,孙云汉,刘晓平,等.冬虫夏草及人工虫草菌丝抗Lewis肺癌的研究.中药通报,1987,12(2):205
[37]董竞成,董晓辉.黄芪注射液与白细胞介素2增强树突细胞抗肿瘤转移作用的比较研究.中国中西医结合杂志,2005,25(3):236-239
[38]Mitani N,Murakanfi K,Yamaura T,et al.Inhibitory effect of berberine on the mediastinal lymph node metastasis produced by orthotopic implantation of Lewis lung carcinoma.Cancer Lett,2001,165(1):35-42
[39]孔庆志,黄涛,黄冬生,等.苦参素对小鼠移植性S180肉瘤血管形成的抑制作用.中国药师,2003,6(12):769-771
[40]冯刚,孔庆志,黄冬生,等.薏苡仁注射液对小鼠移植性S180肉瘤血管形成的抑制作用.肿瘤防治研究,2004,31(4):229-230
[41]薛东波.人参皂苷Rg3抗肿瘤转移作用的研究进展.中国中西医结合外科杂志,2001,7(4):289-290
[42]陈明伟,倪磊.人参皂苷Rg3对肿瘤血管生长调控因子蛋白表达抑制作用的研究.中国中药杂志,2005,30(5):357-360
[43]沃兴德,丁志山.姜黄素及其衍生物抑制肿瘤作用的实验研究.浙江中医学院学报,2005,29(2):53-58
[44]冯敢生,李欣.中药白及提取物抑制肿瘤血管生成机制的实验研究.中华医学杂志,2003,83(5):412-416
[45]丁怡,赵庆亮,李勇刚,等.雷公藤甲素对血管内皮细胞迁移活性的作用.四川大学学报(医学版),2005,36(3):347-350
[46]张前.羟基红花黄色素A抑制新生血管形成的机制研究.北京中医药大学学报,2004,27(3):25-29
[47]江蔚新,蒋燕,季字彬,等.补中益气汤对EAC小鼠癌细胞LPO含量的影响.黑龙江医药,1997,10(2):88-90
[48]林洪生,李树奇,朴炳奎,等.三参冲剂对肺癌转移中内皮细胞及黏附因子的影响.中国肿瘤,1999,8(12):574-576
[49]王彦刈,吴爱姣,许楠,等.复方丹参对高转移性人肺癌细胞与血管内皮细胞黏附及黏附分子表达的影响.中国现代应用药学杂志,2003,5(20):343-345
[50]许建华,范忠泽,孙钰等.肠胃清抗结肠癌转移的机制研究.中国中医药科技,2006,13(3):148-150
[51]许继平,裘维焰,应栩华,等.扶肺煎对原发性肺癌抗转移的临床研究.中国中医药信息杂志,2000(8):40-41
[52]王希胜,张英.山仙颗粒对小鼠黑色素瘤肺转移中nm23/NDPK水平的影响.陕西中医,2006,27(2):244-245
[53]叶向荣,张丹,赵飞.加味血府逐瘀汤抗癌细胞转移的实验研究.山东中医药大学学报,2000,24(6):464-465
[54]陈达理,张绪慧.鳖甲煎丸抗肿瘤血管生成的实验研究.浙江中医杂志,2004,12(12):535-537
[55]徐振晔,王中奇.肺岩宁对晚期非小细胞肺癌生长转移和血清VEGF的影响.上海中医药大学学报,2003,17(3):18-22
[56]王笑民,王征.固本消瘤胶囊抑制小鼠lewis肺癌生长及抗血管生成研究.中国实验方剂学杂志,2004,10(5):50-52
[57]施京红,刘德传.清香散对湿热型肝癌模型大鼠细胞凋亡、肿瘤血管生成的影响.陕西中医,2004,25(1):84-86
[58]刘玉琴,高进,李健生.金龙胶囊抗肿瘤复发、转移的实验研究.北京医学.2005,27(9):554-557
[1]De Vita VT,Hellman S,Rosenberg S.Principles of Oncology(5th Edition).Philadelphia:Lippincott Raven Publisher,1997:135
[2]Gwag BJ,Canzoniero LM,Sensi SL,et al.Calciumion ophores can induce either apoptosis or necrosis in cultured cortical neurons.Neuro science,1999,90(4):1339-1348
[3]曹泽毅.激素受体及临床应用.北京:北京医科大学、中国协和医科大学联合出版社,1993:48-70
[4]Rroemer G,Petit P,Zamzam N,et al.The biochemistry of programmed cell death.FASEB J,1995,9:1277
[5]Gottlieb RA,Nordberg J,Skowronski E,et al.Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.Proc Natl Acad Sci USA,1996,93:654
[6]Barry MA,Reynolds JE,and Eastman A.Etoposide induced apoptosis in human HL 260 cells is associated with intracellular acidification.Cancer Res,1993,53:2349
[7]Gottlieb RA,Giesing H,Zhu J,et al.The acid deoxyribonuclease of neut rophils a possible participant in apoptosis associated genome destruction.Blood,1995,86:2414
[8]Melino G,Annicchiarico Petruzzelli M,Piredda L,et al.Tissue transglutaminase and apoptosis:Senseand antisense transfection studies with human neuro blastoma cells.Mol Cell Biol,1994,14:6584
[9]Paddock SW.Principle and practices of laser scanning confocal microscopy.Mol Biotechnol,2000,16(2):127-149
[10]吴明耀,吴贤英,郑瑞明,等.激光扫描共聚焦显微镜观察免疫组化染色标本.临床与实验病理学杂志,2001,17(6):533-534
[11]司徒镇强,吴军正.细胞培养.西安:世界图书出版西安公司,2007:200
[12]王珊,周春喜,李宁.检测早期细胞凋亡的流式细胞术.现代科学仪器,2003,3:48-50.
[13]赵婷秀,姜霞,王秀莲,等.激光共聚焦显微镜观察FITC-Annexin V/PI双染色法检测人肺癌细胞凋亡.湖北中医学院学报,2005,7(4):9-11
[14]刁志宏,张明霞,朱幼芙.乙型肝炎病毒P22e蛋白对人肝细胞癌细胞株HepG2凋亡的影响.南方医科大学学报,2007,27(11):1649-1652
[15]袁兰.激光扫描共聚焦显微镜技术教程.北京:北京大学医学出版社,2004:79.
[16]王敏,Vogel I,Kalthoff H.尿激酶型纤溶酶原激活因子及抑制剂表达与人大肠癌细胞转移能力的关系.实用肿瘤杂志,2002,17(2):112-114
[17]余琛,洪有采,张慧,等.一种高灵敏度的小檗碱高效液相色谱分析方法.色谱,1994,12(1):37-38
[18]金镇,裴凌,王德智.肿瘤坏死因子对脐静脉血管内皮细胞增殖和细胞内游离钙浓度的影响.中国妇产科杂志,2000,35:657-659
[19]Kroemer G,Petit P,Zamzami N,et al.The biochemistry of programmed cell Death.FASEB J,1995,9(13):1277
[20]Zhu WH,Loh TT.Effects of Na~+/H~+ antiport and intracellular pH in the regulation of HL- 60 cell apoptosis.Biochem Biophys Acta,1995,1269(2):122
[21]KaiserN,Edelman IS.Calicium depedence of glucocorticoid-induced Lymphocytolysis.Proc Natl Acad Sci USA,1977,74:6382642
[22]Squier M KT,Miller ACK,Malkinson AM.Calpain activation in apoptosis.J Cell Physiol,1994,159:2292237
[23]Barry MA,Eastman A.Identification of deoxyribonuclease as an endonuclease involved in apoptosis.Arch Biochem Biophys,1993,300(1):440
[24]Matsubara K,Kubota M,Adachi S,et al.Different mode of cell death by calcium ionophore in human leukemia cell lines:possible role of constitutive endomuclease.Exp Cell Res,1994,210(1):19