【摘要】 目的克隆甲胎蛋白(AFP)、survivin(SUR)、人端粒酶逆转录酶(hTERT)基因启动子,检测并评价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AFP、SUR、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,检测AFP、SUR、hTERT基因启动子在肝癌细胞系和正常组织中的转录活性,评价其转录活性水平和肿瘤特异性。结果成功克隆了AFP、SUR、hTERT启动子,证实AFP、SUR、hTERT启动子在肝癌细胞中具有肿瘤特异性转录活性,SUR启动子活性最高,其活性水平为AFP启动子的52~98倍;hTERT启动子次之,其活性水平为AFP启动子的14~30倍;AFP启动子活性最低。结论 hTERT和SUR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。更多还原

【Abstract】 Objective To clone the AFP,survivin and hTERT gene promoters and to detect their transcriptional activities in various hepatocellular carcinoma cell lines and in human fibroblast cells.And to make groundwork for the targeting gene therapy for human hepatocellular carcinoma.Methods The fragment of AFP,survivin and hTERT promoters were acquired by PCR amplification and cloned into the reporter plasmid pGL3-Basic,containing a luciferase gene.The constructed eukaryotic expression plasmid was transfected into three HCC cell lines and the fibroblast cells.At 24 h post transfection(p.t.),the activity of the luciferase was determined with Dual-Luciferase Reporter Assay System.Results The AFP,survivin and hTERT promoter were successfully cloned into the eukaryotic reporter vector,pGL3-Basic and gained the pGL3-AFP,pGL3-SUR and pGL3-hTERT vector.The AFP,survivin and hTERT promoters had high transcriptional activities in all the three HCC cell lines and no transcriptional activities in the fibroblast cells.Transcription activity detection showed that all of them possessed some tumor-spesific transcription activity and surviving and hTERT promoter showed much higher activity than the AFP promoters.Conclusion The survivin and hTERT promoter possess the tumor-spesific high transcrioptional activity in all three established HCC cell lines.They may serve as useful tool for transcriptional targeting of HCC gene therapy.更多还原