AZT对肝癌细胞端粒酶活性及相关蛋白表达的影响  

Effects of 3'-Azido-deoxythymidine on Telomerase Activity and Protein Expression of Hepatocarcinoma Cell Line SMMC-7721

作　　者：何敏[1] 蒋玉艳[2] 朱淼[1] 韦霄[2] 覃健[2] 张志勇[2] 李力[3] 

机构地区：[1]广西医科大学医学科学实验中心,广西南宁530021 [2]广西医科大学公共卫生学院环境卫生学教研室,广西南宁530021 [3]广西壮族自治区肿瘤医院,广西南宁530021

出　　处：《癌症》2006年第5期543-548,共6页Chinese Journal of Cancer

基　　金：国家自然科学基金项目(No.30460121);广西科学基金项目(No.0135008;No.0322025-2;No.0428005-12)~~

摘　　要：背景与目的:端粒酶抑制剂抑制端粒酶活性的机制十分复杂,可能涉及多个蛋白的共同作用。本研究通过3′-叠氮脱氧胸苷(3′-azido-deoxythymidine,AZT)作用于人肝癌细胞SMMC-7721,比较AZT作用后癌细胞端粒酶活性及相关蛋白的变化,探讨AZT抑制端粒酶活性的可能机制。方法:利用噻唑蓝[3(4,5-demethylthiazole-2-yl)-2,5-diphenyltetrazolium-bromide,MTT]实验确定AZT作用的最佳作用时间和浓度;实时荧光定量端粒重复序列扩增法(real-timefluorescentquantitativeTRAPassay,FQ-TRAP法)检测AZT作用后,SMMC-7721细胞端粒酶活性变化;用缺口末端标记技术(terminaldeoxyribonucleotidyltransferse(TdT)-mediatedbiotin-16-dUTPnick-endlabelling,TUNEL)法和流式细胞术检测AZT作用后,SMMC-7721细胞凋亡情况;单细胞激光拉曼光谱技术和蛋白质芯片-飞行时间质谱仪,检测AZT作用后特异蛋白质的变化。结果:AZT抑制癌细胞生长的最佳作用时间为48h,最佳作用浓度为20mmol/L;FQ-TRAP法检测发现,AZT作用后肝癌细胞端粒酶活性与对照组相比受到明显抑制(P=0.0001);TUNEL法观察到AZT诱导肝癌细胞凋亡形态学改变,流式细胞术检测AZT诱导肝癌细胞凋亡率为13.5%;单细胞激光拉曼光谱技术观察到,AZT作用后有7个与蛋白代谢相关的特征峰变化;蛋白质芯片-飞行时间质谱仪检测发现,AZT作用后有24个蛋白分子在癌细胞中高表达,8个蛋白分子在癌细胞中低表达,32个差异蛋白均属于小分子蛋白。结论:AZT可抑制SMMC-7721细胞端粒酶活性,诱导凋亡与相关特异小分子蛋白有关。BACKGROUND ＆ OBJECTIVE： The mechanism of inhibiting telomerase activity by telomerase inhibitors is very complex, and involves common actions of many proteins. This study was to investigate the effects of 3＇-azido-deoxythymidine （AZT） on telomerase activity and protein expression of hepatocarcinoma cell line SMMC-7721, and explore possible mechanism of inhibiting telomerase activity in SMMC-7721 cells by AZT. METHODS： Optimized concentration and treatment time of AZT were detected by MTT assay. After treatment with AZT, the telomerase activity in SMMC-7721 cells was detected by real-time fluorescent quantitative TRAP （FQ-TRAP） assay; the apoptosis of SMMC-7721 cells was detected by TUNEL assay and flow cytometry （FCM）; the changes of specific proteins were monitored by raman spectra assay and surface-enhanced laser desorption time of flight-mass spectrum （SELDI-TOF-MS） with protein chip assay. RESULTS： The optimized treatment time of AZT was 48 h, and the optimized concentration of AZT was 20 mmol/L. When treated with 20 mmol/L AZT for 48 h, the telomerase activity in SMMC-7721 cells was inhibited by 53.85% of control （P〈0.001）; apoptotic morphologic changes of SMMC-7721 cells were observed; the apoptosis rate of SMMC-7721 cells was 13.5%; 7 spectral peak of proteins were changed; 24 proteins were up-regulated and 8 were down-regulated in SMMC-7721 cells. All of the 32 distinct proteins were small molecular proteins. CONCLUSION. AZT can inhibit the telomerase activity and induce apoptosis of SMMC-7721 cells, which is related to specific small molecular proteins.

关 键 词：3'-叠氮脱氧胸苷 肝肿瘤 SMMC-7721细胞 端粒酶活性 拉曼光谱 蛋白芯片 

分 类 号：R735.7[医药卫生—肿瘤]