Ad-VEGFP-CD/TK系统诱导胃癌细胞凋亡的实验研究

目的 探讨腺病毒介导的VEGF启动子驱动的CD/TK双自杀基因体系(Ad-VEGFP-CD/TK)对胃癌细胞SGC-7901的体外杀伤作用.方法 用重组腺病毒Ad-VEGFP-CD/TK体外感染表达VEGF的SGC-7901细胞,荧光显微镜观察其感染效率,然后给予前药GCV和5-FC,用光镜、透射电镜观察及流式细胞术等方法观察细胞凋亡、细胞周期、细胞内DNA含量及钙离子浓度的变化.结果 腺病毒的感染率随病毒滴度的增高而递增,在感染复数为100时,前药GCV和5-FC在一定浓度搭配范围内呈剂量和时间依赖性地诱导SGC-7901细胞凋亡,电镜下可见细胞凋亡的典型改变.用流式细胞仪测定用药组出现典型的凋亡峰;细胞周期分析显示治疗后细胞G0-G1期比率增多,G2-M及S期细胞减少;此外,前药还可以引起细胞内Ca2 浓度持续增加.结论 VEGF启动子可调控双自杀基因系统选择性诱导表达VEGF的SGC-7901细胞凋亡,其机制与细胞内钙离子浓度升高有关.

Effect of VEGF promoter-driven recombinant adenovirus containing double suicide genes on apoptosis of human gastric carcinoma cells

Objective To study the effect of the adenovirus containing CD/TK fusion gene controlled by the human vascular endothelial growth factor(VEGF)promoter on apoptosis of human gastric carcinoma cells SGC-7901.Methods VEGF-expressing SGC-7901 cells were infected by the recombinant adenovirus Ad-VEGFP-CD/TK,and the infection efficiencies were observed with fluorescence microscopy.The toxic effect and intracellular calcium concentration induced by 5-fluorocytosine(5-FC)and ganciclovic(GCV)were determined by light microscopy,electron microscopy and flow cytometry.Results The transfection efficiency of the recombinant adenovirus in SGC-7901 cells increased with the viral titer.At the multiplicity of infection(MOI)of 100,5-FC and GCV could induce apoptosis of SGC-7901 cells within a given dose range in a dose-and time-dependent manner,and apoptotic changes of the cells were observed with electron microscopy.Apoptotic peak was also detected by flow cytometry.Cell cycle analysis revealed increased cell percentage in G0-G1 phase and decreased percentage of cells in G2-M and S phases in response to treatment with the pro-drugs,which also induced marked elevation of intracellular calcium concentration in the infected cells.Conclusions CD/TK fusion gene system driven by VECF promoter selectively induces apoptosis of VEGF-expressing SGC-7901 cells,the action of which is probably mediated by intracellular calcium variation.