人端粒酶启动子调控双自杀基因治疗肝癌的实验观察  

Transcriptional targeting gene therapy of double suicide gene driven by hTERT promoter in hepatic carcinoma

作　　者：范文哲[1] 杨建勇[1] 陈伟[1] 黄勇慧[1] 王于[1] 张应强[1] 李家平[1] 

机构地区：[1]中山大学附属第一医院肿瘤介入科,广州510080

出　　处：《中华医学杂志》2011年第43期3080-3085,共6页National Medical Journal of China

基　　金：广东省科技计划项目（20098030801027）;广东省自然科学基金（7001663）

摘　　要：目的探讨人端粒酶逆转录酶的核心启动序列（hTERT）启动子驱动的双自杀基因系统CDglyTK真核质粒对肝癌细胞的特异性杀伤作用。方法利用PCR扩增hTERT启动子、SV40、yCD及TKgly基因片段，单独或共同连至pEGFP—CI质粒载体，分别构建pEGFP—hTERT-CD、pEGFP-hTERT—TK及pEGFP—hTERT—CDglyTK质粒，并转染SMMC7721肝癌细胞和HL7702正常人肝细胞，观察其转染效率并以RT—PCR、QPCR、Western印迹方法检测目的基因的表达，给予不同浓度的5-Fc及更昔洛韦（GCV）处理，在用MTT法评估杀伤效应和旁观者效应，TRAP-银染法检测杀伤前后细胞端粒酶活性，流式细胞仪检测细胞凋亡率，比较和分析联合基因与单基因疗效的差异。结果最终质粒的酶切产物所得片段与预期一致，RT—PCR、QPCR及Western印迹结果显示目的基因CD、TK及CDglyTK高表达；pEGFP—hTERT-CD、pEGFP-hTERT—TK及pEGFP—hTERT—CDglyTK转染效率分别为74．5％、76．3％、76．9％。转染pEGFP—hTERT—CDglyTK的细胞较单基因转染对前药具有更高的敏感性（P=0．020，P=0．015）、促细胞凋亡作用（P=0．023，P=0．017）及旁观者效应（P=0．012，P=0．001），可更大程度降低端粒酶活性（P=0．045，P=0．038）。HL7702细胞的转染及生长未受自杀基因系统的影响。结论本研究构建的hTERT启动子驱动的双自杀基因（CDglyTK）系统质粒，在体外实验中对人肝癌细胞具有特异性的高杀伤作用。Objective To examine the selective killing effects of pEGFP-Cl-mediated double suicide gene system driven by the hTERT promoter （hTERT-CDgIyTK） on hepatic carcinoma cells. Methods The bTERT promoter and gene fragments SV40, yCD and TKgly were amplified by PCR （polymerase chain reaction） and then inserted into pEGFP-C1. And the constructs of pEGFP-hTERT-CD, pEGFP-hTERT-TK and pEGFP-hTERT-CDgIyTK were transfected to SMMC 7721 or HL7702 respectively. The transfectional effects were observed and the cellular expressions of suicide genes detected by RT-PCR （reverse transeription-polymerase chain reaction）, QPCR （quantitative polymerase chain reaction） and Western blot. The transfected cells were treated with 5-fluorocytosinc and ganclelovir at different concentrations and the cell-killing and bystander effects evaluated by the method of MTT （3-（4,5）-dimethylthiahiazo （-z-yl）- 3,5-di-phenytetrazoliumromide）. The activity of cell telomerase was detected by the method of TRAP- argentation and the apoptotic rates analyzed by flow cytometry. All results of double and single gene systems were analyzed. Results The fragments of enzyme digestion corresponded to the expectations. RT-PCR, QPCR and Western blot demonstrated the expressions of CD, TK and CDg]yTK. pEGFP-hTERT-CD, pEGFP-hTERT-TK and pEGFP-hTERT-CDglyTK showed the similar transfection efficiencies in SMMC7721 （74. 5% , 76. 3% , 76.9% ）. More sensitive to the prodrugs （ P=0. 020, P=0. 015 ） , higher apoptotic rates （P =0. 023, P =0. 017） and bystander effects（P =0. 012, P =0. 001 ）and lower telomerase activities （ P = 0. 045, P = 0. 038 ） were observed in double gene system versus those in single gene system. However,the transfection and growth of HL7702 cell could not be infected by this double suicide gene. Conclusion The plasmid of CDglyTK fusion gene system driven by hTERT promoter has been successfully constructed. It has demonstrated highly specific killing effects on hepatic carcinoma cells.

关 键 词：肝肿瘤 端粒 末端转移酶 自杀基因 

分 类 号：R735[医药卫生—肿瘤]