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目的探讨运用增强子增强hTERT启动子转录活性后,调控双自杀融合基因CDTK载体对人宫颈癌HeLa细胞的体外杀伤作用。方法将SV40增强子、CMV增强子、CMV增强子/启动子及SV40-CMV双增强子分别与hTERT启动子组合构建成报告基因载体,转染HeLa细胞后用双荧光素酶系统分析其活性差异;然后构建pHr-SCT/CDTKG治疗载体转染HeLa细胞,用流式细胞仪检测转染效率,RT-PCR检测融合自杀基因mRNA表达,HPLC检测5-FU浓度,MTT分析载体杀瘤的效果。结果HeLa细胞中增强子能使hTERT启动子活性提高6～13倍,其中SV40-CMV双增强子/hTERT启动子的活性最高,达到CMV增强子/启动子的近3倍;体外转染pHr-SCT/CDTKG后可检测到CDTK的mRNA的表达;细胞上清中5-FU最高浓度为50.83ug/mL;当5-FC浓度为200ug/mL、GCV浓度为10ug/mL时,HeLa细胞的相对细胞存活率为48.7%。结论SV40-CMV双增强子联合hTERT启动子能高效靶向的调控CDTK融合自杀基因杀伤宫颈癌细胞,因而是一种很有前景的基因治疗宫颈癌的策略。
OBJECTIVE: To investigate the in vitro cytotoxicity of double suicide fusion gene CDTK vector on human cervical carcinoma HeLa cells after enhancing the transcriptional activity of hTERT promoter by using enhancer. Methods SV40 enhancer, CMV enhancer, CMV enhancer / promoter and SV40-CMV double enhancer were respectively combined with hTERT promoter to construct a reporter vector. After transfecting HeLa cells, the luciferase system was used to analyze the difference in activity Then the constructed vector was transfected into HeLa cells. The transfection efficiency was detected by flow cytometry. The mRNA expression of fusion suicide gene was detected by RT-PCR. The concentration of 5-FU was detected by HPLC. The killing effect was evaluated by MTT assay. Results The enhancer of HeLa cells increased the hTERT promoter activity by 6 ~ 13 times, and the SV40-CMV double enhancer / hTERT promoter had the highest activity, nearly 3 times that of CMV enhancer / promoter. The in vitro transfection of pHr- The expression of CDTK mRNA was detected after SCT / CDTKG; the highest concentration of 5-FU in the cell supernatant was 50.83ug / mL; when the concentration of 5-FC was 200ug / mL and GCV concentration was 10ug / mL, the relative expression of HeLa cells Cell survival rate was 48.7%. Conclusion SV40-CMV dual enhancer combined with hTERT promoter can effectively target CDTK fusion suicide gene to kill cervical cancer cells, which is a promising strategy for gene therapy of cervical cancer.