腺病毒驱动KDR启动子介导CD/TK融合基因系统对肝癌细胞的靶向杀伤作用

目的 研究腺病毒介导的KDR启动子驱动CD/TK双自杀基因系统（AdKDR- CDglyTK）对肝癌细胞选择性杀伤作用。方法 将质粒pAdEasy-KDR-CDglyTK在293细胞内包装、扩增后，体外感染表达KDR的BEL-7402细胞株和对照组不表达KDR的LS174T细胞株，并给予不同浓度的前药5-FC和/或GCV，观察该体系对不同细胞株的杀伤效应及其旁观者效应。结果 所得病毒滴度为2.5×10^12pfu/ml。两种细胞的感染率相似，其感染率随腺病毒滴度的递增而增加。RT-PCR方法 检测发现：感染AdKDR-CDglyTK的BEL-7402有目的 基因CDglyTK的表达，感染AdKDR- CDglyTK的LS174T细胞无目的 基因表达。表达KDR的BEL-7402细胞对前药具有较高的敏感性，不表达KDR的LS174T细胞对前药不敏感（F=750.03，P〈0.001）。融合基因的疗效优于任一单自杀基因（F=275.89，P〈0.05）。将感染腺病毒的细胞与未感染细胞以不同比例混合培养，观察到该体系明显的旁观者效应。结论 KDR基因启动子可调控双自杀基因系统选择性杀伤表达KDR的肝癌细胞。

Adenovirus mediated fusion gene system driven by KDR promoter kills selectively liver cancer cells

Objective To evaluate the selectively killing effect of adenovirus (Ad) mediated double suicide gene driven by KDR promoter on liver cancer cells. Methods In this study, 293 packaging cells were transfected by plasmids of pAdEasy-KDR-CDgly TK and the infectious virus was generated in the cells. KDR producing ceils of BEL-7402 and the KDR nonproducing cells of LS174T were infected by the virus respectively, followed by pro-drug 5-FC and/or GCV treatment. The killing and bystander effects on the ceils were analyzed using KDR nonproducing LS174T as controls and targets. Results The infection rate of the recombinant Ad to all the cells was not different. The infected ceils exhibited different sensibility to the two prodrugs: BEL-7402 cells infected with Ad-KDR-CDglyTK were highly sensitive to the prodrugs, but the LS174T cells infected with Ad-KDR-CDglyTK were not sensitive to the two prodrugs (P<0. 001). The killing effect of CD/TK fusion gene on the BEL-7402 cells was stranger than each suicide gene alone (P<0. 05), significant bystander effect was observed on the virus noninfected ceils when eoeultured with the virus infected cells. Conclusions The CD/TK fusion gene system controlled by KDR promoter has selectively killing effect on BEL-7402 liver cancer cells.