hTERT启动子的克隆及在胃癌细胞中的转录活性研究

Clone of the hTERT Promoter and Its Specific Transcriptional Activity in Human Gastric Caner Cells

目的:克隆人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)核心启动子,研究hTERT启动子在人胃癌细胞SGC7901和正常人成纤维细胞HLF中的转录活性。方法:以Hela细胞的基因组DNA为模板,PCR扩增hTERT核心启动子片段,将其克隆入荧光素酶报告基因质粒pGL3-Basic构建hTERT-pGL3Basic表达载体。将该载体用脂质体转染法转染SGC7901和HLF细胞,检测hTERT启动子在这两种细胞中的转录活性。结果:成功克隆hTERT核心启动子;双酶切和PCR鉴定均显示hTERT-pGL3Basic表达载体构建成功;荧光素酶活性检测显示hTERT启动子在SGC7901细胞中的转录活性为阳性对照的21.5%,而在HLF细胞中无活性。结论:hTERT启动子具有肿瘤特异性,可以用于肿瘤的靶向基因治疗。

Objective： To clone a human telomerase reverse transcriptase （hTERT） core promoter, and to investigate the transcriptional activity of the hTERT promoter in the human gastric cancer cells SGC7901 and the normal human fibroblast cells HLF. Methods： The hTERT core promoter was amplified by PCR using the total genomic DNA of Hela cells as sample DNA and then cloned into pGL3-Basic vector. The recombinant was named hTERT-pGL3Basic. This vector was transfected into SGC7901 cells with high telomerase activity and into HLF cells without telomerase activity to detect the transcriptional activities of the hTERT promoter by cationic liposome. Results： The hTERT core promoter was amplified by PCR successfully. The results of restriction enzyme digestion and PCR indicated that the hTERT-pGL3Basic expression vector was constructed successfully as the design. Considering transcriptional activity of pGL3-control in each cell line as 100%, luciferase assay showed the relative transcriptional activity of hTERT promoter in SGC7901 was 21.5% and that ofhTERT promoter in HLF cells was only 0.40%. Conclusions： The hTERT promoter has tumor specificity and may be useful in targeting gene therapy for tumor.