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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。
AIM: To investigate the effect of vascular endothelial growth factor (VEGF) siRNA on proliferation and apoptosis of human renal cell carcinoma cell line (ACHN). Methods: Small interfering RNA (siRNA) against VEGF was chemically synthesized and transfected into ACHN by lipofectamine. The expression of VEGF in the cells was detected by Western blotting. The cell growth curve was determined by trypan blue exclusion assay. MTT colorimetric assay Cell proliferation inhibition rate (IR) was detected, and apoptosis rate (AR) was detected by TUNEL method. Results: The growth curve showed that compared with the blank control group and the negative control group, the growth of ACHN cells in siRNA1 group and siRNA2 group was significantly slowed down. The proliferation inhibition rate of siRNA1 was 10.6%, 18.0 at 24 h, 48 h and 72 h %, 27.1% respectively. The inhibition rates of siRNA2 were 18.9%, 32.7% and 40.3%, respectively, which were higher than that of blank control group and negative control group (P <0.05). The apoptosis rates of siRNA1 group were 10.7%, 15.2% and 20.3 %. The apoptosis rates of siRNA2 group were 17.3%, 26.2% and 37.4%, respectively, which were higher than that of blank control group and negative control group (P <0.05). The expression of VEGF protein in siRNA1 group and siRNA2 group was significantly lower than that in blank control group And negative control group. The inhibitory effect of siRNA2 on IR, AR and VEGF protein expression in ACHN cells were significantly higher than that in siRNA1 group (P <0.05). CONCLUSION: VEGF plays an important role in the development of renal cell carcinoma. Chemically synthesized VEGF-siRNA can specifically inhibit the expression of VEGF in ACHN cell lines and inhibit cell proliferation and apoptosis. For patients with high-expression of VEGF gene in patients with renal cell carcinoma, RNAi technology for VEGF is expected to become a new gene therapy for renal cell carcinoma.