【摘要】 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。更多还原

【Abstract】 [Objective] To investigate the in vitro effects of CDTK/5-FC+GCV on cervix cancer cell line,HeLa cells with an enhanced human hTERT promoter vector system.[Methods] Six transcriptional regulatory elements were constructed by appending a CMV or SV40 enhancer or CMV enhancer/promoter 5’ to the hTERT promoter and further appending a SV40 enhancer to the CMV enhancer/hTERT promoter.Enhanced hTERT promoter activity was determined by luciferase assay after these plasmids were transfected into HeLa cells.Eukaryotic expression vector,pHr-SCT/CDTKG containing a CDTK fusion suicide gene under the control of hTERT promoter combined with the SV40-CMV double enhancers was constructed and transfected into HeLa cells by electroporation.Transfection efficiency was counted by flow cytometry.The mRNA expression of CDTK gene was detected by RT-PCR.Following the transfection,5-FC and GCV were added and high performance liquid chromatography(HPLC) method was applied to determine the level of 5-FU in the supernatant of the transfected cells.Methyl thiazolyl tetrazolium(MTT) assay was used to investigate the anti-tumor effects of pHr-SCT/CDTKG/5-FC+GCV on HeLa cells.[Results] The augmentations of the hTERT promoter activity by those enhancers were increased from 6 to 13 times.Among them the SV40-CMV double enhancers/hTERT promoter reached the highest level and their transcription activity was as nearly 3 times as the CMV enhancer/promoter which was popular used.Also,the CDTK fusion suicide gene was successfully transcribed confirmed by RT-PCR after the transfection.The highest concentration of 5-FU in supernatant of the cells was 92.73 ug/ml.And the relative cell survival rate achieved 48.7% when 5-FC was at 200 ug/mL and GCV was at 10 ug/mL.[Conclusion] The targeting and efficient expression of the fusion suicide gene CDTK regulated by the SV40-CMV double enhancers/hTERT promoter could effectively kill Hela cells in vitro,which can be a promising strategy of gene therapy for cervix cancer.更多还原