增强型自杀基因载体治疗宫颈癌的体外实验研究  

In vitro research of enhanced suicide gene vector for cervix cancer therapy

作　　者：王丽娜[1] 张雅坤[1] 周银[1] 李剑[1] 尹彪[1] 李琼[1] 潘乾[1] 薛志刚[1] 夏昆[1] 梁德生[1] 夏家辉[1] 

机构地区：[1]中南大学中国医学遗传学国家重点实验室,湖南长沙410078

出　　处：《中国医学工程》2006年第6期567-570,575,共5页China Medical Engineering

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。[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 ceils. 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 ceils by electroporation. Transfection efficiency was counted by flow cytometry. The mRNA expression of CDTK gene was detected by RT-PCR. Following the transfeetion, 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 ceils. Methyl thiazolyl tetrazolium （MTr） assay was used to investigate the anti-tumor effects of pHr-SCT/CDTKG/5-FC＋GCV on HeLa ceils. [Results] The augmentations of the hTERT promoter activity by those enhancers were increased from 6 to 13 times. Among them the SV40-CMV double enhaneers/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.

关 键 词：HTERT启动子 增强子 基因治疗 宫颈癌 

分 类 号：Q782[生物学—分子生物学] R737.33[医药卫生—肿瘤]