KDR启动子驱动的双自杀基因对人肝癌细胞及脐静脉内皮细胞的特异性杀伤作用

A double suicide gene system driven by KDR promoter selectively kills human hepatic carcinoma cells and human umbilical vein endothelial 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对人肝癌细胞及脐静脉内皮细胞的特异性杀伤作用。方法用腺病毒AdKDR-CDglyTK感染表达KDR的BEL-7402、HUVEC细胞和不表达KDR的HepG2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。结果以感染复数MOI为100的腺病毒AdKDR-CDglyTK分别感染各细胞株,重组腺病毒对三种细胞具有相似的感染效率。RT-PCR检测发现,重组腺病毒及感染AdKDR-CDglyT的三种细胞均有目的基因CDglyTK的表达。感染腺病毒的BEL-7402、HUVEC细胞对前药具有较高的敏感性,而HepG2细胞对前药不敏感(P<0.001)。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。结论KDR启动子驱动的双自杀基因系统CDglyTK对肝癌细胞及人脐静脉内皮细胞具有特异性的杀伤作用。

Objective To study the selective killing effects of adenovirus （Ad）-mediated double suicide gene system driven by KDR promoter （KDR-CdglyTK） on the human hepatic carcinoma cells and human umbilical vein endothelial cells （HUVECs）. Methods KDR-expressing BEL-7402 and HUVECs and HepG2 cells that did not express KDR were infected by KDR-CdglyTK, and the infection efficiency and the expression of CdglyTK in the cells was detected by RT-PCR. The infected cells were treated with the the prodrugs 5-FC and GCV at different concentrations, and the cell-killing effects and bystander effects were evaluated by MTT method. Results At the multiplicity of infection （MOI） of 100, the recombinant AdKDR-CDglyTK showed similar infection efficiency in the 3 cell lines. RT-PCR demonstrated CDglyTK expression in the recombinant adenovirus and the 3 infected cell lines. BEL-7402 and HUVECs infected by the KDR-CdglyTK, but not the HepG2 cells, were highly sensitive to the prodrugs （/9〈0.001）. Bystander effects of the double suicide gene system were observed in the coculture of the infected and non-infected BEL-7402 and HUVECs. Conclusion The double suicide gene system driven by KDR promoter has specific killing effect on KDR-expressing hepatocellular carcinoma cells and HUVECs.