siRNA沉默VEGF基因对肾癌细胞增殖与凋亡的影响

目的：探讨siRNA干扰血管内皮生长因子（VEGF）基因对人肾细胞癌细胞株（ACHN）细胞增殖与凋亡的影响.方法：化学合成针对VEGF的小干扰RNA,通过脂质体转染至ACHN中,利用Western印迹法检测细胞内VEGF的表达,采用台盼蓝拒染法测定细胞生长曲线,用MTT比色分析法检测细胞增殖抑制率（IR）,用TUNEL方法检测细胞凋亡率（AR）.结果：生长曲线提示,与空白对照组及阴性对照组相比,siRNA1组、siRNA2组ACHN细胞的生长明显减慢 在24 h、48 h、72 h,siRNA1的增殖抑制率为10.6%、18.0%、27.1%,siRNA2增殖抑制率为18.9%、32.7%、40.3%,均高于空白对照组及阴性对照组（P〈0.05） siRNA1组的细胞凋亡率为10.7%、15.2%、20.3%,siRNA2组的细胞凋亡率为17.3%、26.2%、37.4%,均高于空白对照组及阴性对照组（P〈0.05） siRNA1组、siRNA2组VEGF蛋白表达水平明显低于空白对照组及阴性对照组,其中siRNA2对ACHN细胞的IR、AR和VEGF蛋白表达的抑制作用均显著高于siRNA1组（P〈0.05）.结论：VEGF在肾癌的发生发展中起着重要作用,化学合成的VEGF-siRNA能特异性抑制肾细胞癌ACHN细胞株中VEGF的表达,抑制细胞生长增殖,促进细胞凋亡.对于VEGF基因高表达的肾细胞癌患者,针对VEGF的RNAi技术有望成为肾细胞癌新的基因治疗手段.

Effects of Small Interfering RNAs Targeting VEGF on the Proliferation and Apoptosis of Renal Cell Carcinoma Line ACHN

Objective： To study the influence of small interfering RNA of vascular endothelial growth factor （VEGF） on the cell proliferation and apoptosis in renal cell carcinoma line ACHN. Methods： ACHN cells were transfected with small interfering RNAs （siRNAs） by liposome transfection targeting against the human VEGF by chemosynthesis,the cell growth curves determined by trypan blue staining, The inhibition of proliferation of ACHN cell was studied by methyl thiazolyl tetrazolium （MTT） assay,cell apoptosis detected byTUNEL assay. The expression of VEGF in the levels of protein was detected by Western blot method. Results： The cell growth curves showed the growth of ACHN cells of blank control and negative control were suppression significantly（P〈 0. 05）. The inhibition rate（IR） of siRNA1 group（ 10.6 , 18.0, 27. 1 ） and siRNA2 group（ 18. 9 , 32. 7%, 40. 3） were significantly higher than those in the blank control group and negative control group respectively at 24 h,48 h,72 h（P〈0. 05）. The apoptosis index of siRNA1 group（10. 7,15.2,20. 3） and siRNA2 group （17.3 ,26.2 ,37.4 % ） were obviously higher than those in the blank control group and negative control group respectively at 24 h,48 h,72 h（P〈0. 05）. The cells of siRNA1 ,siRNA2 group transected with a specific VEGF： siRNA showed suppression of VEGF protein expression compared with those of he blank control group and nega-？ tire control group. Moreover, the effects of siRNA2 on inhibition rate, apoptosis index and down-regulating ex- pression of VEGF protein were stronger than the group of siRNA1 （P〈O. 05＇）. ConcluslonstVEGF plays an important role in the carcinogenesis and development of renal cell carcinoma , siRNAs targeting against human VEGF by chemosynthesis may specially suppress the expression of VEGF protein in renal cell carcinoma cells, inhibit cell prolil＇eration and enhance cell apoptosis. The RNAi targeting VEGF may become as a novel gene therapeutic strategy for VEGF overexpressed renal cell carcinoma.