【摘要】 为了探讨恶性肿瘤基因治疗的新靶点,本研究以vegfr-2的mRNA为靶点,设计、构建vegfr-2 shRNA的真核表达载体,用脂质体法将重组质粒转染人非小细胞肺癌A549细胞,用RT-PCR法及Western blot分别检测重组质粒对该基因mRNA和蛋白表达的抑制效果,应用CCK-8法检测细胞增殖、抑制情况,Hoechst染色法检测细胞凋亡形态学变化。结果表明:与空白对照、空质粒对照及scramble对照相比,干扰组shRNA能有效地降解vegfr-2mRNA,尤其转染pGenesil-1-vegfr-2-shRNA-1质粒组内源性vegfr-2 mRNA及蛋白减少更显著;在不同时间点转染干扰质粒组的A值均降低,转染后72小时抑制率最高,干扰组与空质粒组和空白对照组相比,对细胞生长抑制作用的差异有统计学意义(p<0.01);Hoechst荧光染色显示,转染48小时后在荧光显微镜下干扰组细胞呈典型的细胞凋亡形态。结论:vegfr-2 shRNA真核表达载体可序列特异性下调A549细胞的基因转录和表达,有效抑制细胞增殖和诱导凋亡,提示VEGF/VEGFR-2信号通路可作为恶性肿瘤基因治疗和功能研究的理想靶点。更多还原

【Abstract】 This study was aimed to explore a novel potential gene taget for therapy of malignant tumor.The recombinant expression plasmids of VEGF/VEGFR-2 were designed,constructed and then transfected into A549 cells by using lipofectamine.The expressions of VEGF/VEGFR-2 mRNA and protein were detected by RT-PCR and Western blot respectively.The cell proliferation was assayed by CCK-8 and the cell apoptosis was detected using Hoechst Staining.The results indicated that as compared with the blank control,pGenesil-1 and scramble groups,the interference effect of pGenesil-1-vegfr-2-shRNA-1 vector group was more obvious.As the expression of endogenous vegfr-2 mRNA decreased,the expression of VEGFR-2 protein decreased correspondingly.The proliferation of A549 cells was inhibited significantly by RNAi at 72 hours(p<0.01).The apoptosis of A549 cells was induced at 48 hours after being transfec-ted with pGenesil-1-vegfr-2-shRNA-1 and the typical apoptosis morphology could be seen by fluorescence microscopy.It is concluded that the expression of vegfr-2 gene is inhibited effectively by vegfr-2 specific shRNA.The proliferation of A549 cells is inhibited significantly and the apoptosis of cells is induced.This result showed that VEGF/VEGFR-2 signaling pathway can be an effective taget for the prevention and treatment of malignant tumor.更多还原