【摘要】 目的:探讨shRNA抑制C-erbB-2基因表达对小鼠肺腺癌Lewis细胞凋亡的影响。方法:构建4个pGPU6/RFP/Neo-erbB-2质粒和pGPU6/RFP/Neo-shNC。应用Lipofectamine 2000转染pGPU6/RFP/Neo-shNC至小鼠肺腺癌Lewis细胞,应用荧光显微镜观察荧光,确定最佳转染效率。应用RT-PCR检测C-erbB-2 mRNA的水平,应用Western blotting检测该蛋白的表达,筛选干扰效果最好的质粒。应用流式细胞术检测空白组、pGPU6/RFP/Neo-shNC组和最佳干扰组细胞的凋亡率。结果:pGPU6/RFP/Neo-shNC(μg):Lipofectamine(μl)为0.4:1时,转染效果最好。4个pGPU6/RFP/Neo-erbB-2质粒均能降低C-erbB-2 mRNA水平和蛋白的表达,其中干扰效果最好的是pGPU6/RFP/Neo-erbB-2-mus-3669,用于后续实验。pGPU6/RFP/Neo-erbB-2-mus-3669质粒组的凋亡率高于空白组和pGPU6/RFP/Neo-shNC组(P<0.05)。结论:pGPU6/RFP/NeoerbB-2-mus-3669质粒能抑制小鼠Lewis细胞C-erbB-2基因的表达,诱导细胞凋亡。更多还原

【Abstract】 Objective: To investigate the effect of C-erbB-2 gene inhibition by shRNA on the apoptosis of Lewis cells. Methods: 4 pGPU6/RFP/Neo-erbB-2 plasmid and pGPU6/RFP/Neo-shNC were synthesized.pGPU6/RFP/Neo-shNC was transfected into mouse Lewis cells to test the transfection efficiency. The interf-erence effect was tested by RT-PCR and Western blotting to select the best effective C-erbB-2 shRNA. The apoptotic rate of cells was tested by FCM. Results: The transfection efficiency was the best when pGPU6/RFP/Neo-shNC(μg): Lipofectamine(μl) was 0.4:1. pGPU6/RFP/Neo-erbB-2-mus-3669 had the best interference effect, and it could induce higher apoptotic rate of Lewis cells than control group and pGPU6/RFP/Neo-shNC group(P < 0.05). Conclusion: pGPU6/RFP/Neo-erbB-2-mus-3669 can inhibit the expression of C-erbB-2 gene and induce the apoptosis of Lewis cells.更多还原