慢病毒介导的CD/TK基因靶向杀伤胃癌细胞的作用

Lentivirus-mediated CD/TK gene selectively kills human gastric adneocarcinoma cells

目的:探讨慢病毒介导的血管内皮细胞生长因子受体(KDR)启动子驱动的CD/TK双自杀基因体系(FGW-KDRP-CD/TK)抑制胃癌SGC-7901细胞的体外杀伤作用.方法:用重组慢病毒FGW-KDRP-CD/TK体外感染表达KDR的SGC-7901细胞,荧光显微镜观察其感染效率;RT-PCR,WesternBlot方法检测转基因细胞CD/TK的表达;用细胞计数法绘制细胞生长曲线;用流式细胞术观察用药后细胞周期的变化.给予前药更昔洛韦(GCV)和5-氟胞嘧啶(5-FC)处理后,采用MTT法观察该体系对SGC-7901细胞杀伤效应及其旁观者效应.结果:慢病毒的感染率随病毒滴度的增高而递增.经RT-PCR,West-ernBlot检测发现转基因细胞有目的基因及蛋白产物的表达.从细胞生长曲线可以看出已转染慢病毒SGC-7901和未转染SGC-7901细胞增殖情况相似,其差异无显著性.流式细胞术检测结果与对照相比较,随着前药剂量的增大,处于S期细胞明显减少.MTT法检测结果显示前药呈剂量依赖性抑制SGC-7901细胞生长.同时该体系存在明显的旁观者效应.结论:KDR启动子可以调控融合基因体系选择性地杀伤人胃癌SGC-7901细胞,并存在旁观者效应.

AIM：To study the killing effect of lentivirus-mediated CD/TK double gene controlled by kinase insert domain-containing receptor（KDR）promoter on human gastric carcinoma cells SGC-7901 in vitro.METHODS：SGC-7901 cells（with KDR expression）were transfected with FGW-KDRP-CD/TK vector in vitro.The infection efficiency in SGC-7901 cells was observed under fluorescence microscope.And the expression of CD/TK in genetically modified cells was detected by RT-PCR and Western Blot.The cell growth curve was drawn according to cell counts.Flow cytometer（FCM）was used for cell cycle analysis in the treatment group.Followed by treatment of the ganciclovir（GCV）and 5-fluorocytosine（5-FC）,the killing and bystander effects of suicide gene therapy system on SGC-7901 cells were evaluated by MTT method.RESULTS：The transfection efficiency in SGC-7901 cells increasedwith the increasing lentiviral titer.RT-PCR and Western Blot demonstrated that there existed the CD/TK gene and the protein gene product in genetically modified cells.No significant difference was found in the characteristics of growth between the transfected SGC-7901 cells and the non-transfected cells with lentiviral vector according to cell growth curve.Flow cytometry analysis showed the proportion of S phase cells decreased in the treatment group compared with the control group as increasing the prodrug concentration.Prodrug could inhibit proliferation of SGC-7901 cells and the effect was dose-dependent by MTT analysis.In addition,a considerable bystander effect was also observed in the suicide gene system.CONCLUSION：CD/TK fusion gene system driven by KDR promoter can selectively kill SGC-7901 cells,and there exists an obvious bystander effect during this process.