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目的探讨血管内皮生长因子siRNA对恶性黑素瘤细胞株A375增殖和凋亡的影响。方法构建针对血管内皮生长因子的siRNA真核表达载体pU-VEGF-siRNA将其表达载体经电穿孔转染A375细胞,采用逆转录聚合酶链反应(RT-PCR)、免疫酶联吸附实验(ELISA)方法检测转染后的A375细胞VEGF的mRNA和蛋白表达,应用细胞计数比较转染组和对照组A375细胞增殖能力,将含转染组和对照组A375细胞的上清液分别培养人静脉内皮细胞(ECV-304),观察其生长情况,并用流式细胞仪观察转染pU-VEGF-siRNA载体对A375细胞凋亡的影响。结果转染pU-VEGF-siRNA载体后,A375细胞的VEGFmRNA和蛋白表达均较对照组明显下降,并且其生长缓慢,其细胞周期出现亚二倍凋亡峰。用转染pU-VEGF-siRNA的A375细胞上清液培养的ECV-304细胞增殖力较对照组下降。结论通过RNA干扰技术阻断VEGF的表达,可抑制A375和ECV-304细胞增殖,诱导A375细胞凋亡。
Objective To investigate the effects of vascular endothelial growth factor (siRNA) on the proliferation and apoptosis of malignant melanoma cell line A375. Methods The eukaryotic expression vector pU-VEGF-siRNA targeting VEGF was transfected into A375 cells by electroporation. The expression of VEGF was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay ) Method was used to detect the mRNA and protein expression of VEGF in transfected A375 cells. The proliferation of A375 cells in the transfected group and control group was compared by cell counting. The supernatant of A375 cells containing transfected and control cells were cultured in human vein endothelial (ECV-304). The growth of A375 cells was observed. The effect of pU-VEGF-siRNA vector on the apoptosis of A375 cells was observed by flow cytometry. Results After transfection with pU-VEGF-siRNA vector, the expression of VEGFmRNA and protein of A375 cells were significantly decreased compared with the control group, and its growth was slow. The sub-doubling apoptotic peak appeared in the cell cycle. The proliferation of ECV-304 cells cultured in the supernatant of A375 cells transfected with pU-VEGF-siRNA was lower than that of the control group. Conclusion Blocking the expression of VEGF by RNA interference can inhibit the proliferation of A375 and ECV-304 cells and induce the apoptosis of A375 cells.