人端粒酶逆转录酶干扰增强肿瘤坏死因子相关的凋亡诱导配体诱导肝癌细胞凋亡的机制  

Mechanisms of human telomerase reverse transcriptase RNAi which increases hepatocellular carcinora cell apoptosis induced by TRAIL

作　　者：张汝钢[1] 房殿春[1] 宁晓燕[1] 王国安[1] 

机构地区：[1]第三军医大学西南医院全军消化病研究所,重庆400038

出　　处：《中华肝脏病杂志》2007年第1期32-36,共5页Chinese Journal of Hepatology

基　　金：全军“十五”科研基金（01MA172）

摘　　要：目的探讨人端粒酶逆转录酶（hTERT）干扰增加肿瘤坏死因子相关的凋亡诱导配体（TRAIL）诱导肝癌HepG2和SMMC7721细胞凋亡的分子机制。方法采用膜联蛋白V／碘化丙锭染色的流式细胞术方法检测细胞凋亡；采用Western blot方法检测凋亡相关蛋白Procaspase-8、-9、-3及Bax、Bcl-2和hTERT表达；采用端粒重复扩增法和端粒数量和长度测定法检测端粒酶活性和端粒长度。结果hTERT干扰显著增加TRAIL诱导的肝癌细胞凋亡。100ng／ml TRAIL作用24h后，HepG2细胞凋亡率由5．53％增加至10．35％；SMMC 7721细胞凋亡率由14．73％增加至77．24％。hTERT干扰明显增加Procaspase-8、-9和Bcl-2表达，显著降低Bax表达，明显促进TRAIL作用后Procaspase-8、-9、-3活化，并且hTERT干扰后端粒酶活性显著降低，端粒长度明显缩短，然而对照细胞与未转染细胞相比各指标均无明显变化。结论hTERT干扰明显增加TRAIL诱导的肝癌细胞凋亡，其机制可能与Procaspase-8、-9表达增加，端粒酶活性降低和端粒长度缩短有关，而与Bcl-2和Bax表达无关。Objective To investigate the mechanisms for human telomerase reverse transcdptase （hTERT） RNA interference （RNAi） in increasing hepatocellular carcinoma cell apoptosis induced by TNF- related apoptosis-inducing ligand （TRAIL）. Methods Cell apolStosis was identified by flow cytometry analysis after annexin V/PI double staining. Expression of apoptosis-related proteins, procaspase-8, -9, -3, Bax, Bcl-2 and hTERT, were identified by Western blotting analysis; telomerase activity and telomere length were detected by telomeric repeat amplification protocol （TRAP） and telomere amount and length assay （TALA） methods. Results Hepatocellular carcinoma cell apoptosis induced by TRAIL were all significantly increased by hTERT RNAi （P 〈 0.05）. For example, apoptosis rates were enhanced from 5.53% （untransformed） to 10.35% （transformed） in HepG 2 cells and from 14.73% to 77.24% in SMMC 7721 cells after being treated by 100 ng/ml TRAIL for 24 h. Moreover, activation of procaspase-8, -9 and -3 in transformed cells after being treated by TRAIL were all significantly raised （P 〈 0.05） in a dose-dependent manner. The expression of procaspase-8, -9 and Bcl-2 were effectively augmented （P 〈 0.05）, but expressions of Bax and hTERT were strikingly decreased （P 〈 0.05）. Meanwhile, telomerase activity was apparently suppressed and telomere length was markedly shortened （P 〈 0.05）. There were no remarkable differences in these effects between control cells and the untransformed cells （P 〉 0.05）. Conclusion Enhanced cell apoptosis induced by TRAIL through hTERT RNAi may be related to up-regulation of procaspase-8 and -9 expressions. However the down-regulation of hTERT expression, reduced telomerase activity and shortened telomere length may not be related to expressions of Bcl-2 and Bax.

关 键 词：癌 肝细胞 细胞凋亡 RNA干扰 逆转录酶 人端粒 

分 类 号：R686[医药卫生—骨科学]