端粒酶逆转录酶(hTERT)启动子的克隆及在人肝癌细胞的肿瘤特异性分析  

Cloning of human telomerase reverse transcriptase gene promoter and analysis of specific activities in human hepatocellular carcinoma cells

作　　者：况舸[1] 黄爱龙[1] 唐霓[1] 

机构地区：[1]重庆医科大学病毒性肝炎研究所,重庆400010

出　　处：《重庆医科大学学报》2005年第3期325-328,共4页Journal of Chongqing Medical University

基　　金：863项目资助(资助号:2001AA217121)教育部感染性疾病分子生物学重点实验室

摘　　要：目的:克隆hTERT基因启动子并检测其在不同肝癌细胞系和正常组织细胞中的转录活性,为肝癌靶向性基因治疗提供依据。方法:采用PCR法扩增获得hTERT基因的启动子片段,将之克隆到表达虫荧光素酶基因的报告质粒上,通过测定荧光素酶报告基因的表达,检测hTERT基因启动子在肝癌细胞系和正常组织中的转录活性,并将之与甲胎蛋白(alpha—fetalprotein,AFP)基因启动子活性相比较,评价其作为肝癌基因治疗中应用的肿瘤特异性启动子的可行性。结果:成功克隆了hTERT启动子,证实hTERT启动子在肝癌细胞中具有相当强的转录活性,其活性水平为AFP启动子的14～30倍;在原代培养的成纤维细胞中其启动子未表现转录活性。结论:hTERT启动子在肝癌细胞系中具有较强的启动活性和显著的肿瘤特异性,可望开发成为新型的肝癌靶向性基因治疗工具。Objective:To clone the hTERT gene promoter and detect its transcriptional activities in various hepatocellular carcinoma cell lines and in human fibroblast cells in order to make groundwork for the targeting gene therapy for human hepatocellular carcinoma.Methods:The fragment of hTERT promoters were acquired by PCR amplification and were cloned into the reporter plasmid pGL3-Basic which contains a luciferase gene.The constructed eukaryotic expression plasmid pGL3-hTERT in which the expression of the luciferase were drived by hTERT promoter were transfected into three HCC cell lines and the primary fibroblast cells.At 24 hours post transfection (p.t.),the activity of the luciferase was determined with Dual-Luciferase Reporter Assay System.A pGL3-AFP containing luciferase gene controlled by the AFP promoter was used as positive control and a pGL3-Basic vector which have no promoter in front of the luciferase gene was used as negative control.Results:The hTERT promoter was successfully cloned into the eukaryotic reporter vector,pGL3-Basic and gained the pGL3-hTERT vector.The hTERT promoters showed high transcriptional activities in all the three HCC cell lines but no transcriptional activities in the primary fibroblast cells.The transcriptional activity of the hTERT promoter was much higher than AFP promoter(14～30 times).Conclusion:Our data revealed that the hTERT promoter possesses the tumor-spesific high transcrioptional activity in all the three established HCC cell lines.It may serve as a useful tool for transcriptional targeting of hepatocellular carcinoma gene therapy.

关 键 词：基因表达 人端粒酶逆转录酶 基因疗法 肝癌 

分 类 号：R575[医药卫生—消化系统]