以KDR为启动子的双自杀基因系统诱导结肠癌细胞凋亡的实验研究

摘要

目的 研究以KDR为启动子的双自杀基因系统CDglyTK诱导结肠癌细胞凋亡的实验研究.方法 用联合基因AdKDR-CDglyTK腺病毒转染表达KDR不同的人结肠癌细胞HCT116和LS174T,检测转染效率;RT-PCR检测双自杀基因CDglyTK的表达;用梯度浓度的前药5-FC及GCV处理,MTT法检测双自杀基因系统对HCT116和LS174T的细胞毒性作用;流式细胞术观察细胞凋亡变化;免疫组织化学法检测caspase-3蛋白的表达.结果 携带双自杀基因的重组腺病毒载体成功转染结肠癌细胞.RT- PCR结果显示:表达KDR的HCT116细胞能表达目的 基因,而不表达KDR的LS174T细胞不表达目的 基因.5-FC和GCV对转染腺病毒的HCT116有明显的细胞毒作用而对LS174T细胞不敏感.流式细胞仪结果显示:给予5-FC和GCV后HCT116细胞出现凋亡峰,G0/G1升高、S期下降.Caspase-3表达增强(P＜0.01).结论 KDR启动子驱动的双自杀基因系统CDglyTK能特异性杀伤结肠癌HCT116细胞,KDR可作为结肠癌基因治疗的靶点,并通过增强caspase-3途径诱导细胞凋亡.%Objective To study the apoptosis effect induced by a double suicide gene system driven by the KDR promoter (KDR-CDglyTK)on the colon carcinoma cells. Methods Adcnovirus (Ad)-mcdiatcd fusion gene system (KDR-CDg1yTK)on the colon carcinoma infected HCT116 and LS174T cells that express KDR was different. The infection efficiency and the expression of CDglyTK in the cells were detected by RT-PCR. The infected cells were treated with the 5-FC and GCV at different concentrations and the cytotoxic effect by MTT method. Apoptosis in HCT116 cells was detected by flow cytomctry. The expression of caspasc-3 was studied by immunocytochcmical technique. Results Rccom-binant adcnovirus vector carried double suicide gene successfully transfected colon cancer cells. RT-PCR results showed that HCT116 cells expressed KDR can express purpose gene,the LS174T cells no expressed KDR did not express purpose gene. 5-FC and GCV showed obvious cell poison effect on HCT116 cells and did not have cell poison effect on LS174T cells. Flow cytomctry showed a apoptotic peak and increase for cell percentage in G0-G1 phase and decrease for percentage in S phase in HCT116 cell. The expression of caspasc-3 was incrcascd(P<0. 01). Conclusion CDglyTK fusion gene system driven by the KDR promoter can specific damage HCT116 colon cancer cells. KDR can be used as colon cancer gene therapy targets. KDR-CDglyTK could induce apoptosis through the enhancement caspase 3 ways.