KDR启动子驱动双自杀基因诱导胃癌细胞凋亡的实验研究

目的探讨KDR启动子驱动双自杀基因体系对胃癌细胞凋亡的诱导作用。方法以重组腺病毒AdEasy-KDR-CDglyTK体外感染表达KDR的SCG7901细胞株和不表达KDR的HepG2细胞株，并给予不同浓度的前药GCV和（或）5-FC，观察该体系对SCG7901细胞的杀伤效应。分别应用透射电镜、Hoechest33258／PI染色、流式细胞仪观察细胞超微结构、细胞凋亡率和细胞周期的变化。结果携带双自杀基因（CDglyTK）和报告基因（GFP）的重组腺病毒载体转染后，95％以上SCG7901和HepG2细胞中有GFP表达。表达KDR的SCG7901细胞对前药具有较高的敏感性，不表达KDR的HepG2细胞对前药不敏感（P〈0．01）。两种前药联合应用优于任一前药单独应用的疗效（P〈0．01）。流式细胞术检测表明该体系可抑制SCG7901细胞DNA的合成，表现为s期细胞比例增高及G2期细胞减少。同时，Hoechest33258／PI染色和电镜下可见SCG7901有凋亡改变。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SCG7901细胞，并且该体系可以诱导胃癌细胞凋亡。

Experimental study on the apoptosis of gastric cancer cells induced by adenovirus mediated fusion gene system driven by KDR promoter

Objective To study the selective tumoricidal effect of adenovirus (Ad)-mediated fusion gene system driven by kinase domain-containing (KDR) promoter on human stomach adenocarcinoma SCG7901 cells, and to observe its apoptosis induction effect. Methods Both the AdEasy-KDR-CDglyTK cells with KDR-expressing SCG7901 cells and non KDR-expressing HepG2 cells were reconstructed, and then they were treated with the pro-drugs 5-FC and/or GCV at different concentrations. The killing effects of the transfection on the cells were evaluated. Flow cytometry was employed to determine the cell cycle distribution, and Hooechest/PI stain and electron microscopy performed to observe the pathological changes of cells. Results The green fluorescent protein (GFP) was observed in 95% of the infected SCG7901 and HepG2 cells when the multiple of infection (MOI) of the Ads was 100. No significant difference on the growth feature was found among the transfected SCG7901, HepG2 and untransfected SCG7901 and HepG2 cells. The infected cells exhibited different sensitivities to the two pro-drugs: SCG7901 cells infected with rAd were highly sensitive to the pro-drugs, while the infected HepG2 cells were not (P<0.01). The killing effect of CDglyTK fusion gene on the target cells was much stronger than that of either single suicide gene (P<0.01). In addition, the cell cycle of SCG7901 cells was arrested at S phase with morphologic feature of apoptosis displayed by electron microscopy. Conclusion KDR promoter may regulate the CDglyTK fusion gene system to selectively kill the KDR-expressing SCG7901 cells and to induce the cell apoptosis.