目的 :探讨VP2 2的细胞间传递作用对HSV tk/GCV杀伤肿瘤细胞的促进作用。方法 :将VP2 2与报告基因 绿色荧光蛋白基因 (GFP)或前药酶基因 单纯疱疹病毒胸苷激酶基因 (HSV tk)构建在同一载体上 ,进行DNA序列分析鉴定 ;将此融合基因载体转染 2 93T或COS7细胞 ,免疫荧光分析确定转染情况和细胞间传递作用 ;在前药GCV不同浓度、转染与未转染细胞比例不同时 ,MTT法检测细胞增殖情况 ;利用转染细胞培养液上清培养未转染细胞 ,确定旁观者效应是否由培养液转移。结果 :PCR及序列分析显示融合基因插入载体正确 ;对 2 93T或COS7细胞转染率可达 2 5 %～ 3 0 % ,且证明融合蛋白已被表达并在细胞间传递 ;HSV tk与VP2 2融合后在前药GCV浓度低至 0 .1μg/ml时就可显示出细胞杀伤效应增强 ,并随着表达VP2 2 HSV tk细胞比例的增加 ,细胞杀伤作用增强。结论 :VP2 2介导的自杀基因产物的细胞间传递作用可解决自杀基因的低效细胞毒作用 ,明显增强细胞杀伤效应。 Objective: To investigate the role of VP2 2 in cell-to-cell transmission in HSV tk / GCV killing tumor cells. Methods: VP2 2 was constructed on the same vector as the green fluorescent protein gene (GFP) of the reporter gene or the herpes simplex virus thymidine kinase gene (HSV tk) of the prodrug enzyme gene, and was identified by DNA sequence analysis. The fusion gene vector was transfected 2 93T or COS7 cells were detected by immunofluorescence analysis to determine the transfection and cell-cell transfection. MTT assay was used to detect the proliferation of transfected and non-transfected cells at different concentrations of prodrug GCV. Supernatants were cultured in non-transfected cells to determine if bystander effect was transferred from the culture broth. Results: PCR and sequence analysis showed that the insertion of the fusion gene was correct; the transfection rate of 293T or COS7 cells was 25% ~ 30%, and the fusion protein was expressed and transferred between cells; HSV tk and VP2 2 After the fusion, the cytotoxicity was enhanced when the concentration of the prodrug GCV was as low as 0.1 μg / ml, and the cytotoxicity was enhanced as the proportion of VP2 2 HSV tk cells was increased. Conclusion: The intercellular transmission of suicide gene mediated by VP2 2 can solve the inefficient cytotoxic effect of suicide gene and significantly enhance the cytotoxic effect.