11种二萜类化合物对膀胱癌体外抑制作用的初步研究
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摘要
[目的]：膀胱癌是最常见的恶性肿瘤之一,膀胱癌术后复发率高是临床关注的焦点问题,但目前的化疗药物膀胱灌注治疗不能达到满意的疗效。因此,寻找一种安全有效的新型治疗药物迫在眉睫。本课题通过研究11种二萜类化合物对体外培养的人膀胱癌T24细胞抑制作用,从而筛选出新的膀胱癌化疗药物。
[方法]：体外培养人膀胱癌T24细胞,筛选最佳接种浓度。采用四氮甲基唑蓝(MTT)比色法,以丝裂霉素(MMC)作为阳性对照,研究SA-1、S-3、CM-6、S-11、S-4、S-1、CM-4、S-12、CM-3、S-5、SR-37等11种二萜类化合物在不同浓度下对T24细胞生长的抑制作用并测定其半数抑制浓度(IC50)。先用浓度为20μ mol/L的各样品进行初步筛选,根据初筛结果,在该浓度即20μmol/L对肿瘤细胞生长抑制率在50%附近的化合物设5个浓度梯度复筛,分别为40、8、1.6、0.32、0.064μ mo1/L五个浓度梯度,检测24小时及48小时二萜类化合物抑制膀胱癌T24细胞0D值,结合相关公式计算出各化合物的IC50值。
[结果]：MMC、SA-1、S-3、CM-6、S-11、S-4、S-1、CM-4、S-12、CM-3、S-5、SR-37对膀胱肿瘤细胞T24作用48小时的IC50值分别为：2.55、6.16、6.64、8.87、6.06、3.79、8.25、6.69、7.94、8.33、10.46μmol/L。11种样品中有10种对膀胱癌T24细胞增殖IC50值小于10ug/mL。其中二萜类化合物S-4对膀胱癌T24细胞的IC50值最低。
[结论]：11种二萜类化合物中S-4对膀胱癌T24细胞的抑制率最高；11种化合物对膀胱癌T24细胞的抑制率呈时间依赖性和浓度依赖性；通过对11种二萜类化合物体外抗膀胱癌细胞的初步研究,为S-4应用于膀胱癌病人术后灌注治疗打下基础。
bjective:Bladder cancer is one of the most common malignancies. tumor recurrence is a major clinical concern. Unfortunately, current chemotherapies are insufficient in bladder reperfusion.Therefore, there is an obvious urgent need for novel, effective and safe therapies to prevent both recurrence and progression. According to study on effect of11natural diterpenoids to inhibite bladder cancer in vitro, we can screen a new chemotherapy of bladder cancer.
Method:To obtain the best consistency of bladder cancer T24cells in vitro, Using MTT colorimetric method and taking MMC as the positive control to research the inhibitory effects of11antitumor compounds to the bladder cancer in vitro:SA-1、 S-3、CM-6、S-11、S-4、S-1、CM-4、S-12、CM-3、S-5and SR-37on the growth of T24cells and its value of IC50with40、8、1.6、0.32、0.064μmol/L five different concentration in different hour24h and48h. Firstly, taking20μmol/L concentration of each sample as preliminary screening. Based on the result of the preliminary screening, we set40、8、1.6、0.32、0.064μmol/L screen again to detect the OD of11diterpenoids to inhibite the bladder cancer in vitro in24h and48h, then figure up each IC50with relevance formula.
Result:The IC50of MMC. SA-1、S-3、CM-6、S-11、S-4、S-1、CM-4、S-12、 CM-3、S-5、SR-37to the T24bladder cancer cell in48h as follows:2.55、6.16、6.64、8.87、6.06、3.79、8.25、6.69、7.94、8.33、10.46μmol/L. There are10kinds of compounds which values of IC50are less than10ug/mL. S-4has the lowest value of IC50to the T24bladder cancer cells.
Conclusion:Among11compounds. S-4has the highest value of IC50to the T24bladder cancer cells. The inhibition rate of the11compounds to the T24bladder cancer cells assume time dependence and concentration dependence. By preliminary study on inhibitory effect of11natural diterpenoids to bladder cancer in vitro, S-4had layed foundations in treatment of bladder cancer patients.
[方法]：体外培养人膀胱癌T24细胞,筛选最佳接种浓度。采用四氮甲基唑蓝(MTT)比色法,以丝裂霉素(MMC)作为阳性对照,研究SA-1、S-3、CM-6、S-11、S-4、S-1、CM-4、S-12、CM-3、S-5、SR-37等11种二萜类化合物在不同浓度下对T24细胞生长的抑制作用并测定其半数抑制浓度(IC50)。先用浓度为20μ mol/L的各样品进行初步筛选,根据初筛结果,在该浓度即20μmol/L对肿瘤细胞生长抑制率在50%附近的化合物设5个浓度梯度复筛,分别为40、8、1.6、0.32、0.064μ mo1/L五个浓度梯度,检测24小时及48小时二萜类化合物抑制膀胱癌T24细胞0D值,结合相关公式计算出各化合物的IC50值。
[结果]：MMC、SA-1、S-3、CM-6、S-11、S-4、S-1、CM-4、S-12、CM-3、S-5、SR-37对膀胱肿瘤细胞T24作用48小时的IC50值分别为：2.55、6.16、6.64、8.87、6.06、3.79、8.25、6.69、7.94、8.33、10.46μmol/L。11种样品中有10种对膀胱癌T24细胞增殖IC50值小于10ug/mL。其中二萜类化合物S-4对膀胱癌T24细胞的IC50值最低。
[结论]：11种二萜类化合物中S-4对膀胱癌T24细胞的抑制率最高；11种化合物对膀胱癌T24细胞的抑制率呈时间依赖性和浓度依赖性；通过对11种二萜类化合物体外抗膀胱癌细胞的初步研究,为S-4应用于膀胱癌病人术后灌注治疗打下基础。
bjective:Bladder cancer is one of the most common malignancies. tumor recurrence is a major clinical concern. Unfortunately, current chemotherapies are insufficient in bladder reperfusion.Therefore, there is an obvious urgent need for novel, effective and safe therapies to prevent both recurrence and progression. According to study on effect of11natural diterpenoids to inhibite bladder cancer in vitro, we can screen a new chemotherapy of bladder cancer.
Method:To obtain the best consistency of bladder cancer T24cells in vitro, Using MTT colorimetric method and taking MMC as the positive control to research the inhibitory effects of11antitumor compounds to the bladder cancer in vitro:SA-1、 S-3、CM-6、S-11、S-4、S-1、CM-4、S-12、CM-3、S-5and SR-37on the growth of T24cells and its value of IC50with40、8、1.6、0.32、0.064μmol/L five different concentration in different hour24h and48h. Firstly, taking20μmol/L concentration of each sample as preliminary screening. Based on the result of the preliminary screening, we set40、8、1.6、0.32、0.064μmol/L screen again to detect the OD of11diterpenoids to inhibite the bladder cancer in vitro in24h and48h, then figure up each IC50with relevance formula.
Result:The IC50of MMC. SA-1、S-3、CM-6、S-11、S-4、S-1、CM-4、S-12、 CM-3、S-5、SR-37to the T24bladder cancer cell in48h as follows:2.55、6.16、6.64、8.87、6.06、3.79、8.25、6.69、7.94、8.33、10.46μmol/L. There are10kinds of compounds which values of IC50are less than10ug/mL. S-4has the lowest value of IC50to the T24bladder cancer cells.
Conclusion:Among11compounds. S-4has the highest value of IC50to the T24bladder cancer cells. The inhibition rate of the11compounds to the T24bladder cancer cells assume time dependence and concentration dependence. By preliminary study on inhibitory effect of11natural diterpenoids to bladder cancer in vitro, S-4had layed foundations in treatment of bladder cancer patients.
引文
[1]吴阶平主编。吴阶平泌尿外科学[M]济南。山东科学技术出版社,2005,2：921-976.
[2]那彦群,孙光,主编。中国泌尿外科疾病诊断治疗指南2009版[M]北京。人民卫生出版社,2009,9：16-31.
[3]Hall MC, Chang SS, Dalbagni G, et al. Guideline for the management of nonmuscle invasive bladder cancer(stages Ta. T1and Tis):2007 update [J]J Urol, 2007,178(6):2314-2330.
[4]Babjuk M, Oosterlinck W, Sylvester R,et al. EAU Guidelines on Non-Muscle-Invasive Urothelial Carcinoma of the Bladder [J]Eur Urol,2008,54(2): 303-314.
[5]王成勇。羟喜树碱膀胱灌注预防浅表性膀胱肿瘤术后复发136例分析[J]蚌埠医学院学报,2010,35(10)：1034-1035.
[6]Di Lorenzo G, Perdona S. Damiano R, Faiella A, et al. Gemcitabine versus bacille Calmette-Guerin after initial bacille Calmette-Guerin failure in non-muscle-invasive bladder cancer:a multicenter prospective randomized trial. [J]Caneer.2010, 116(8):1893-1900.
[7]Herr HW, Laudone VP, Whltmore WF, Jr. An overview of intravesical therapy for superficial bladder tumors [J]. J Urol,1987,138(6):1363-8.
[8]Huncharek M, McGarry R, Kupelnick B. Impact of intravesieal chemotherapy on recurrence rate of recurrent superficial transitional cell carcinoma of the bladder:results of a meta-analysis [J]. Anticancer Res,2001,21(1B):765-9.
[9]Momann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays [J]. J Immunol M eth,1983, 65:55.
[10]韩晓凤,王艳婷,欧阳仁荣。MTT法体外药敏试验研究进展[J].肿瘤,1999,19(1)：55-58.
[11]徐建明,宋三泰,汤仲明等。MTT法体外药敏试验指导下的乳腺预见性化疗[J].中华肿瘤杂志,1997,19(2)：153-156。
[12]王艳萍,姜曙,陈晓禾等。50例神经胶质瘤体外化疗药物敏感性研究[J].四川肿瘤防治,2000,13(3)：142-144.
[13]辛华雯,吴笑春,李罄等.胃癌体外化疗药物敏感性及临床疗效研究[J].中国临床药理学与治疗学,2002,7(2)：147-149.
[14]姜浩,曹建国,杨林等,琼脂培养MTT法测定肺癌和卵巢癌药物敏感性的临床评价[J],湖南医学,2001,18(6)：414-416.
[15]刘昌孝,孙瑞元编著。药物评价实验设计与统计学基础[M)北京.军事医学科学出版社,1999,3：31-32.
[16]徐勇勇主编。医学统计学[M]北京。高等教育出版社,2004第二版,1：18-188.
[17]JemalA., R.Siegel, E. Ward, Y. Hao, J. Xu, T. Murray, M.J. Thun. Cancer statistics,2008[J]. CA Cancer J Clin,2008,58(2):8-29.
[18]Jemal A.,R. Siegel, E. Ward, Y.Hao, J.Xu, T. Murray, M.J. Thun. Cancer statistics,2008[J]. CA Cancer J Clin,2008,58(2):71-96.
[19]Pieter R, Huismans DR, Leyva A,et al. Comparison of the rapid automated MTT-assay with adycex elusion assay for chemo sensitivity testing in child-blood leukaemia. Br J Caneer 1989,59:217-220.
[20]Alexandroff AB, Jackson AM, O'Donell MA, et al. BCG immuneotherapy of bladder cancer:20years on. Lancet,1999,353:1689-1694.
[21]O Donnell MA, De Wolf WC. Bacille Calmette Guerin immunotherapy for superficial bladder cancer. New prospects for an old wathorse. Surg Oncol Clin N Am.1995,4(2):189-202.
[22]Steg A, Adjiman S, Debre B. BCG therapy in superficial bladder tumors complications and precautions[J]. Eur Urol,1992,21 Suppl2:35-40.
[23]Pansadoro V, Em-miliozi P, de Paula F, et al. Long-term follow-up of G3TI transi-tional cell carecinoma of the bladder treated with intravesical Bacille Calmette Guerin:18-year experience[J]. Urology,2002,59(2):227-231.
[24]Marks LS, Dipaola RS, Nelson P, et al. PC-SPES:herbal formulation for prostate cancer[J]. Urology,2003,61(6):1292-1300.
[25]Ikezoe T, Chen SS, Tong XJ, et al. Oridonin induces growth inhibition and apopto-sis of a variety of human cancer cells [J]. Int J Oncology,2003,23: 1187-1193.
[26]Wen JM, Sun LB, Zhang M, et al. A non-isotopic method for the detection of telomerase activity in tumor tissues:TRAP-Silver staining assay.J Clin Patllol,1998,51:110-112.
[27]Dalla Torre CA, Maeiel RMB, Pinheiro NA, et al.TRAP-Silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines. BrazJ Med Biol Res,2002,35:65-68.
[28]Husehtscha LI, Jeitner TM, Andersson CE, et al. Identification of apoptotic and neerotic human Leukemic eells by flow cytometry[J]. Exp Cells Res, 1994,212(1):161-165.
[29]Vercanllmen D, Beyaert R, Denecker G, et al. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis faetor[J]. J Exp Med,1998,187(9):1477-1485.
[30]Chan DW, Son SC, Block W, et al. Purification and charaeterization of ATM from human placenta[J]. J Biol Chem,2000,275(11):7803-7810.
[31]Ethardt P, CooPer GM. Activation of the CPP32 apoptotic Protease by distinct signaling pathways with differential sensitivity to Bcl-xl[J].J Biol Chem, 1996,271(30):17601-17604.
[32]Thornberry NA, Lazebnik Y. Caspases:enemies within [J]. Seienee, 1998,281(5381):1312-1316.
[33]Biswas DK, Martin KJ, McAlister C, et al. Apoptosis caused by ehemotherapeutic inhibition of nuclear factor-kappa B activation[J]. Caneer Res, 2003,63(2):290-295.
[34]Fernandes-Alneniri T, Litwaek G, Alnemri ES. CPP32, a novel human apoptotic protein With homology to Caenothabditis elegans cell death protein Ced-3 and marrllnalian inierleukin-1beta-eonverting enzyme[J]. J Biol Chem,1994, 269(49):30761-30764.
[35]Tormanen-Napankangas U, Soini Y, Kahlos K, et al. ExPression of Caspases-3,-6 and-8 and their relation to apoptosis in non-small cell lung carcinoma[J]. Int J Cancer,2001,93(2):192-198.
[36]Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts[J]. Nature,1990,345:458-460.
[37]Kim NW, Piatyszek MA, Prowse KR, et al.Specific association of human telomerase activity with immortal cells and cancer[J].Scienee,1994,266:2011-2015.
[38]Hiyama K, Hiyama E, Ishioka S, et al. Telomerase activity in small-cell and non small-cell lung cancers[J]. J Natl Inst,1995,87:895-902.
[39]Oshinsky GS, ChenY, Jarrett T, el al. A model of bladder tumor xenografts in the nude rat. [J]. J Urol,1995,154:1925-1927.
[40]Bisson JF, Parache RM, Droulle P, el al. A new method of implanting orthotopic rat bladder tumor for experimental therapies. [J]. Int J Cancer,2002,102: 280-282.
[41]Abbaskhan A, choudhary M1, Tsuda Y, et al. A new diepoxyent kauranoid, rugosinin from Isodon rugosus. [J]. Plata Med,2003,69(1):94-96.
[42]Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to Proliferartion and cytotoxicity assays [J] J. Immund Meth ods,1983, 65(1-2):55-63.
[43]张翠卿.唐东平.肿瘤体外药敏试验与临床优化治疗方案的研究进展[J]四川肿瘤防治,2006,19(4)：280-289.
[44]陆艳.MTT法测定肿瘤细胞对化疗药物的敏感性[J]医药导报,2010,29(8)：1002-1005.
[45]陈艳华,黎丹戎,侯华新等.不同溶剂和受试物颜色对MTT实验的影响[J].广西医科大学学报,2007,24(1)：17-19.
[46]辛华主编.现代细胞生物学技术[M]北京.科学出版社,2009第一版,1：95-97、171-172.
[47]Bubenik J, Baresova M, Viklieky V, et al. Established cell line of urinary bladder carcinoma(T24)containing tumor specific antigen [J] Int J Cancer,1973,11 (3):765-773.
[48]Philips BJ, Coyle CH, Morrisror SN, et al. Induction of apoptosis in human bladder cancer cells by green tea catechins[J]. Biomed Res,2009,30(4): 207-215.
[49]Pieter Retal.Comparison of the rapid automated MTT-assay with a dye exclusion assay for chemosensitivity testing in child-blood leukaemia.Br J Caneer 1989,19(5):212-217.
[50]朱慧芬,张悦,王晓林等,MTT比色法检测IL-2和杀伤细胞活性的研究[J].免疫学杂志,1994,10(1)：48-51.
[51]马金霞,潘世扬,张卫,童明庆。MTT比色法用于肿瘤细胞体外药物敏感性试验的检测检测[J].临床检验杂志,2002,20(2)：104.
[52]新药(西药)临床前研究指导原则汇编。中华人民共和国卫生部药政局,1993,137-139.
[1]季宇彬,中药有效成分药理与应用[M]哈尔滨：黑龙江科学技术出版社,1995.
[2]Liu RH. Sun J. Antiproliferative activity of applea is not due to phenolic-induced hydrogen peroxide formation[J]. J Agric Food Chem,2003,51(6): 1718-1723.
[3]SafavyA, Bonner JA, Waksal HW, et al. Synthesis and Biological Evaluation of Paclitaxel-C225 Conjugate as Model for Targeted Drug Delivery(1)[J]. Bioconjug Chem,2003,14(2):302-310.
[4]王玉,黄洁华.紫杉醇联合顺铂治疗晚期卵巢癌的护理[J].护理学杂志,2000,15(7)：409～410.
[5]陈惠祯,谭道彩,吴绪峰主编。现代妇科肿瘤治疗学[M].武汉：湖北科学技术出版社,1998.237.
[6]Sug Hyung Lee, Min Sun Shin, Hong Sug Kim, et al. Somatic mutations of Fas(Apo-1/CD95) gene in cutaneous squamous cell carcinoma arising from a burn scar [J]. The Journal of Investigative Dermatology,2000,114(1): 122-126.
[7]张清媛,康欣梅,赵文辉。紫杉醇联合表阿霉素治疗进展期乳腺癌的近期疗效观察[J].适用肿瘤学杂志,2002,16(3)：216-217.
[8]Fujita Y. Plant Tssue and Cell Culture. New York:Alan R Liss Inc,1987. 169.
[9]LeeK H. Novel antitum or agents from higher plants[J]. Med Res Rev 1999, 19(6):569-596.
[10]Zhang TM, Chen ZY, Chao JH.Study on rube scensine B:a new antitum or procoply isolated from donglincao. Kexue Tongbao,1981:26(10):939-942.
[11]Lin CH, Beale JM, Hurley LH. Anticancer drug design based on plant-derived natural produets [J]. Bioehem,1991,30(3):597-3564.
[12]Zhang Yu, wang Jun, Lou Liguang, et al. Scavenging effect of oridonin on active oxygen free radicals, Henan Medieal Researeh,1999:8(8):100-104.
[13]Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.1983,65:55-63.
[14]Sunsanne G, Ralf T. Bioactive agent from natural source:trends in discovery and application [J]. Advance in Biochemical Engineerin,1999, 6(4):105-154.
[15]樊青霞,李醒亚,王瑞林,等.冬凌草治疗食管癌胃底责门癌287例.中国中西医结合外科杂志,1997：3(6)：380-381.
[16]Wang QD, Liu MJ, Zhang TM. Antiteratogenic and antimutagenic effect of Oridonin. JHnan Med Univ,1992,27(10):120-125.
[17]Zhang TM. Recent status of the study on Rabdosia rubescens. Chin J Oncol, 1982,4:322.
[18]Maron DM, Ames BN. Revised methods for the Sal monella mutagenicy test [J]. Mutat Res,1983,113:173-215
[19]王绵英。冬凌草甲素对小鼠白血病L1210细胞动力学的影响。中国药理 学报.1985,6(3)：195-198.
[20]GilleJ JP, Pasman P, Van Berket CGM, et al. Effect of antioxidants on hyperoxia-induced chromosomal breakage in Chinese hamster ovary cells: protection by carnosine. Mutagenesis,1991; 6:313.
[21]Markert M.Andrews PC, Babiar BM. Measurment of O2 produCtion by human neutrophils. The prepatotion and assay of NADPH oxidase-containing particles from human neutrophils. Thods Enzymol, 1999:105:358
[22]Cragg GM. Newman DJ. Discovery and development of antineoplastic agent from nutural sources[J]. Cancer Invest,1999,17(2):153-163.
[23]Veronese FM. Morpurgo M. Bioconjugation in Pharmaceutieal chemistry[J]. Farmaco,1999,54(8):497-516.
[24]Searle AJF, Willson RL. Stimulation of microsomal lipid peroxidation buy iron and cysteine. Charaeterization and the role of free radicals. Bioehen J, 2000; 2123:549-54.
[25]Lytollis W, et al.5-Alkylresorcinols from Hakea trifurcate That Cleave DNA, J. Am, Chem, Soc,1995,117:12691.
[26]Harvey A L. Mdeicines from nature:are natural products still relevant to drug discovery [J] Trends Pharmacol Sci,1999,20(5):196-198.
[27]Kerr Jf, Winterford CM, Harmon BV. APoPtosis, it significance in cancer and cancer therpy Cancer,1994; 73(8):2013-2026.
[2]那彦群,孙光,主编。中国泌尿外科疾病诊断治疗指南2009版[M]北京。人民卫生出版社,2009,9：16-31.
[3]Hall MC, Chang SS, Dalbagni G, et al. Guideline for the management of nonmuscle invasive bladder cancer(stages Ta. T1and Tis):2007 update [J]J Urol, 2007,178(6):2314-2330.
[4]Babjuk M, Oosterlinck W, Sylvester R,et al. EAU Guidelines on Non-Muscle-Invasive Urothelial Carcinoma of the Bladder [J]Eur Urol,2008,54(2): 303-314.
[5]王成勇。羟喜树碱膀胱灌注预防浅表性膀胱肿瘤术后复发136例分析[J]蚌埠医学院学报,2010,35(10)：1034-1035.
[6]Di Lorenzo G, Perdona S. Damiano R, Faiella A, et al. Gemcitabine versus bacille Calmette-Guerin after initial bacille Calmette-Guerin failure in non-muscle-invasive bladder cancer:a multicenter prospective randomized trial. [J]Caneer.2010, 116(8):1893-1900.
[7]Herr HW, Laudone VP, Whltmore WF, Jr. An overview of intravesical therapy for superficial bladder tumors [J]. J Urol,1987,138(6):1363-8.
[8]Huncharek M, McGarry R, Kupelnick B. Impact of intravesieal chemotherapy on recurrence rate of recurrent superficial transitional cell carcinoma of the bladder:results of a meta-analysis [J]. Anticancer Res,2001,21(1B):765-9.
[9]Momann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays [J]. J Immunol M eth,1983, 65:55.
[10]韩晓凤,王艳婷,欧阳仁荣。MTT法体外药敏试验研究进展[J].肿瘤,1999,19(1)：55-58.
[11]徐建明,宋三泰,汤仲明等。MTT法体外药敏试验指导下的乳腺预见性化疗[J].中华肿瘤杂志,1997,19(2)：153-156。
[12]王艳萍,姜曙,陈晓禾等。50例神经胶质瘤体外化疗药物敏感性研究[J].四川肿瘤防治,2000,13(3)：142-144.
[13]辛华雯,吴笑春,李罄等.胃癌体外化疗药物敏感性及临床疗效研究[J].中国临床药理学与治疗学,2002,7(2)：147-149.
[14]姜浩,曹建国,杨林等,琼脂培养MTT法测定肺癌和卵巢癌药物敏感性的临床评价[J],湖南医学,2001,18(6)：414-416.
[15]刘昌孝,孙瑞元编著。药物评价实验设计与统计学基础[M)北京.军事医学科学出版社,1999,3：31-32.
[16]徐勇勇主编。医学统计学[M]北京。高等教育出版社,2004第二版,1：18-188.
[17]JemalA., R.Siegel, E. Ward, Y. Hao, J. Xu, T. Murray, M.J. Thun. Cancer statistics,2008[J]. CA Cancer J Clin,2008,58(2):8-29.
[18]Jemal A.,R. Siegel, E. Ward, Y.Hao, J.Xu, T. Murray, M.J. Thun. Cancer statistics,2008[J]. CA Cancer J Clin,2008,58(2):71-96.
[19]Pieter R, Huismans DR, Leyva A,et al. Comparison of the rapid automated MTT-assay with adycex elusion assay for chemo sensitivity testing in child-blood leukaemia. Br J Caneer 1989,59:217-220.
[20]Alexandroff AB, Jackson AM, O'Donell MA, et al. BCG immuneotherapy of bladder cancer:20years on. Lancet,1999,353:1689-1694.
[21]O Donnell MA, De Wolf WC. Bacille Calmette Guerin immunotherapy for superficial bladder cancer. New prospects for an old wathorse. Surg Oncol Clin N Am.1995,4(2):189-202.
[22]Steg A, Adjiman S, Debre B. BCG therapy in superficial bladder tumors complications and precautions[J]. Eur Urol,1992,21 Suppl2:35-40.
[23]Pansadoro V, Em-miliozi P, de Paula F, et al. Long-term follow-up of G3TI transi-tional cell carecinoma of the bladder treated with intravesical Bacille Calmette Guerin:18-year experience[J]. Urology,2002,59(2):227-231.
[24]Marks LS, Dipaola RS, Nelson P, et al. PC-SPES:herbal formulation for prostate cancer[J]. Urology,2003,61(6):1292-1300.
[25]Ikezoe T, Chen SS, Tong XJ, et al. Oridonin induces growth inhibition and apopto-sis of a variety of human cancer cells [J]. Int J Oncology,2003,23: 1187-1193.
[26]Wen JM, Sun LB, Zhang M, et al. A non-isotopic method for the detection of telomerase activity in tumor tissues:TRAP-Silver staining assay.J Clin Patllol,1998,51:110-112.
[27]Dalla Torre CA, Maeiel RMB, Pinheiro NA, et al.TRAP-Silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines. BrazJ Med Biol Res,2002,35:65-68.
[28]Husehtscha LI, Jeitner TM, Andersson CE, et al. Identification of apoptotic and neerotic human Leukemic eells by flow cytometry[J]. Exp Cells Res, 1994,212(1):161-165.
[29]Vercanllmen D, Beyaert R, Denecker G, et al. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis faetor[J]. J Exp Med,1998,187(9):1477-1485.
[30]Chan DW, Son SC, Block W, et al. Purification and charaeterization of ATM from human placenta[J]. J Biol Chem,2000,275(11):7803-7810.
[31]Ethardt P, CooPer GM. Activation of the CPP32 apoptotic Protease by distinct signaling pathways with differential sensitivity to Bcl-xl[J].J Biol Chem, 1996,271(30):17601-17604.
[32]Thornberry NA, Lazebnik Y. Caspases:enemies within [J]. Seienee, 1998,281(5381):1312-1316.
[33]Biswas DK, Martin KJ, McAlister C, et al. Apoptosis caused by ehemotherapeutic inhibition of nuclear factor-kappa B activation[J]. Caneer Res, 2003,63(2):290-295.
[34]Fernandes-Alneniri T, Litwaek G, Alnemri ES. CPP32, a novel human apoptotic protein With homology to Caenothabditis elegans cell death protein Ced-3 and marrllnalian inierleukin-1beta-eonverting enzyme[J]. J Biol Chem,1994, 269(49):30761-30764.
[35]Tormanen-Napankangas U, Soini Y, Kahlos K, et al. ExPression of Caspases-3,-6 and-8 and their relation to apoptosis in non-small cell lung carcinoma[J]. Int J Cancer,2001,93(2):192-198.
[36]Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts[J]. Nature,1990,345:458-460.
[37]Kim NW, Piatyszek MA, Prowse KR, et al.Specific association of human telomerase activity with immortal cells and cancer[J].Scienee,1994,266:2011-2015.
[38]Hiyama K, Hiyama E, Ishioka S, et al. Telomerase activity in small-cell and non small-cell lung cancers[J]. J Natl Inst,1995,87:895-902.
[39]Oshinsky GS, ChenY, Jarrett T, el al. A model of bladder tumor xenografts in the nude rat. [J]. J Urol,1995,154:1925-1927.
[40]Bisson JF, Parache RM, Droulle P, el al. A new method of implanting orthotopic rat bladder tumor for experimental therapies. [J]. Int J Cancer,2002,102: 280-282.
[41]Abbaskhan A, choudhary M1, Tsuda Y, et al. A new diepoxyent kauranoid, rugosinin from Isodon rugosus. [J]. Plata Med,2003,69(1):94-96.
[42]Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to Proliferartion and cytotoxicity assays [J] J. Immund Meth ods,1983, 65(1-2):55-63.
[43]张翠卿.唐东平.肿瘤体外药敏试验与临床优化治疗方案的研究进展[J]四川肿瘤防治,2006,19(4)：280-289.
[44]陆艳.MTT法测定肿瘤细胞对化疗药物的敏感性[J]医药导报,2010,29(8)：1002-1005.
[45]陈艳华,黎丹戎,侯华新等.不同溶剂和受试物颜色对MTT实验的影响[J].广西医科大学学报,2007,24(1)：17-19.
[46]辛华主编.现代细胞生物学技术[M]北京.科学出版社,2009第一版,1：95-97、171-172.
[47]Bubenik J, Baresova M, Viklieky V, et al. Established cell line of urinary bladder carcinoma(T24)containing tumor specific antigen [J] Int J Cancer,1973,11 (3):765-773.
[48]Philips BJ, Coyle CH, Morrisror SN, et al. Induction of apoptosis in human bladder cancer cells by green tea catechins[J]. Biomed Res,2009,30(4): 207-215.
[49]Pieter Retal.Comparison of the rapid automated MTT-assay with a dye exclusion assay for chemosensitivity testing in child-blood leukaemia.Br J Caneer 1989,19(5):212-217.
[50]朱慧芬,张悦,王晓林等,MTT比色法检测IL-2和杀伤细胞活性的研究[J].免疫学杂志,1994,10(1)：48-51.
[51]马金霞,潘世扬,张卫,童明庆。MTT比色法用于肿瘤细胞体外药物敏感性试验的检测检测[J].临床检验杂志,2002,20(2)：104.
[52]新药(西药)临床前研究指导原则汇编。中华人民共和国卫生部药政局,1993,137-139.
[1]季宇彬,中药有效成分药理与应用[M]哈尔滨：黑龙江科学技术出版社,1995.
[2]Liu RH. Sun J. Antiproliferative activity of applea is not due to phenolic-induced hydrogen peroxide formation[J]. J Agric Food Chem,2003,51(6): 1718-1723.
[3]SafavyA, Bonner JA, Waksal HW, et al. Synthesis and Biological Evaluation of Paclitaxel-C225 Conjugate as Model for Targeted Drug Delivery(1)[J]. Bioconjug Chem,2003,14(2):302-310.
[4]王玉,黄洁华.紫杉醇联合顺铂治疗晚期卵巢癌的护理[J].护理学杂志,2000,15(7)：409～410.
[5]陈惠祯,谭道彩,吴绪峰主编。现代妇科肿瘤治疗学[M].武汉：湖北科学技术出版社,1998.237.
[6]Sug Hyung Lee, Min Sun Shin, Hong Sug Kim, et al. Somatic mutations of Fas(Apo-1/CD95) gene in cutaneous squamous cell carcinoma arising from a burn scar [J]. The Journal of Investigative Dermatology,2000,114(1): 122-126.
[7]张清媛,康欣梅,赵文辉。紫杉醇联合表阿霉素治疗进展期乳腺癌的近期疗效观察[J].适用肿瘤学杂志,2002,16(3)：216-217.
[8]Fujita Y. Plant Tssue and Cell Culture. New York:Alan R Liss Inc,1987. 169.
[9]LeeK H. Novel antitum or agents from higher plants[J]. Med Res Rev 1999, 19(6):569-596.
[10]Zhang TM, Chen ZY, Chao JH.Study on rube scensine B:a new antitum or procoply isolated from donglincao. Kexue Tongbao,1981:26(10):939-942.
[11]Lin CH, Beale JM, Hurley LH. Anticancer drug design based on plant-derived natural produets [J]. Bioehem,1991,30(3):597-3564.
[12]Zhang Yu, wang Jun, Lou Liguang, et al. Scavenging effect of oridonin on active oxygen free radicals, Henan Medieal Researeh,1999:8(8):100-104.
[13]Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.1983,65:55-63.
[14]Sunsanne G, Ralf T. Bioactive agent from natural source:trends in discovery and application [J]. Advance in Biochemical Engineerin,1999, 6(4):105-154.
[15]樊青霞,李醒亚,王瑞林,等.冬凌草治疗食管癌胃底责门癌287例.中国中西医结合外科杂志,1997：3(6)：380-381.
[16]Wang QD, Liu MJ, Zhang TM. Antiteratogenic and antimutagenic effect of Oridonin. JHnan Med Univ,1992,27(10):120-125.
[17]Zhang TM. Recent status of the study on Rabdosia rubescens. Chin J Oncol, 1982,4:322.
[18]Maron DM, Ames BN. Revised methods for the Sal monella mutagenicy test [J]. Mutat Res,1983,113:173-215
[19]王绵英。冬凌草甲素对小鼠白血病L1210细胞动力学的影响。中国药理 学报.1985,6(3)：195-198.
[20]GilleJ JP, Pasman P, Van Berket CGM, et al. Effect of antioxidants on hyperoxia-induced chromosomal breakage in Chinese hamster ovary cells: protection by carnosine. Mutagenesis,1991; 6:313.
[21]Markert M.Andrews PC, Babiar BM. Measurment of O2 produCtion by human neutrophils. The prepatotion and assay of NADPH oxidase-containing particles from human neutrophils. Thods Enzymol, 1999:105:358
[22]Cragg GM. Newman DJ. Discovery and development of antineoplastic agent from nutural sources[J]. Cancer Invest,1999,17(2):153-163.
[23]Veronese FM. Morpurgo M. Bioconjugation in Pharmaceutieal chemistry[J]. Farmaco,1999,54(8):497-516.
[24]Searle AJF, Willson RL. Stimulation of microsomal lipid peroxidation buy iron and cysteine. Charaeterization and the role of free radicals. Bioehen J, 2000; 2123:549-54.
[25]Lytollis W, et al.5-Alkylresorcinols from Hakea trifurcate That Cleave DNA, J. Am, Chem, Soc,1995,117:12691.
[26]Harvey A L. Mdeicines from nature:are natural products still relevant to drug discovery [J] Trends Pharmacol Sci,1999,20(5):196-198.
[27]Kerr Jf, Winterford CM, Harmon BV. APoPtosis, it significance in cancer and cancer therpy Cancer,1994; 73(8):2013-2026.