KDR启动子驱动的双自杀基因对人结肠癌细胞的特异性杀伤作用

A double suicide gene system driven by KDR promoter selectively kills human colon adneocarcinoma SW480 cells

目的探讨腺病毒介导的KDR启动子驱动的双自杀基因系统CDglyTK对人结肠癌细胞SW480的特异性杀伤作用。方法用腺病毒AdKDR-CDglyTK感染表达KDR的SW480细胞和不表达KDR的LS174T细胞,观察其感染效率并以RT-PCR方法检测重组腺病毒及转基因细胞CDglyTK的表达,然后给予不同浓度的前药5-FC及GCV处理,MTT法检测该系统对不同细胞株的杀伤效应和旁观者效应;用流式细胞术观察细胞内DNA含量和细胞周期的变化。结果携带双自杀基因和报告基因(GFP)的重组腺病毒载体,感染复数为100时,95%以上的受感染SW480和LS174T细胞中有GFP表达。RT-PCR检测发现SW480细胞有目的基因的表达,而LS174T细胞无表达。在前药应用下,已转染腺病毒的SW480和LS174T细胞表现出对前药不同的敏感性:表达KDR的SW480细胞对前药具有较高的敏感性,而不表达KDR的LS174T细胞对前药不敏感(P<0.01)。将感染腺病毒的细胞与未感染细胞以不同比例混合培养,观察到该系统有明显的旁观者效应。用流式细胞仪测定给药组出现典型的凋亡峰,细胞周期显示治疗组细胞G0-G1期比率增多,S期细胞减少。结论KDR启动子可调控双自杀基因体系选择性杀伤人结肠癌SW480细胞,抑制其增殖并诱导细胞凋亡。

Objective To study the selective killing effect of adenovirus（Ad）-mediated double suicide gene system driven by the KDR promoter（KDR-CDglyTK） on human colon adneocarcinoma SW480 cells.Methods KDR-expressing SW480 cells and LS174T cells that did not express KDR were infected by KDR-CDglyTK,and the infection efficiency and the expression of CDglyTK in the cells were detected by RT-PCR.The infected cells were treated with the prodrugs 5-FC and GCV at different concentrations,and the cell-killing effects and bystander effects were evaluated by MTT method.DNA content and the cell cycle changes in SW480 cells were detected by flow cytometry.Results The expression of green fluorescent protein（GFP） was observed in 95% of the infected SW480 and LS174T cells with a multiplicity of infection（MOI） of 100.RT-PCR demonstrated that the product of CD/TK gene existed in SW480 cells infected by Ad-KDR-CD/TK,but not in infected LS174 cells.The infected SW480 cells exhibited high sensitivity to the prodrugs,but the infected LS174T cells did not（P0.01）.Bystander effects of the double suicide gene system were observed in the coculture of the infected and non-infected SW480 cells.At the MOI of 100,treatment of the infected cells with the prodrugs resulted in increased cell percentage in G0-G1 phase and decreased percentage in S phase and the prodrug-treated cells showed an apoptotic peak in flow cytometry.Conclusion CDglyTK fusion gene system driven by the KDR promoter selectively kills and induces the apoptosis of the KDR-CDglyTK SW480 cells.