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目的:探讨干扰素诱导蛋白16(IFI16)siRNA对干扰素-α(IFN-α)诱导的人血管内皮细胞(HVECs)凋亡的影响及其机制。方法:应用转染IFN-α和(或)IFI16siRNA瞬时干预体外培养的HVECs,分别设空白组为阴性对照组,IFN-α加非特异性siRNA转染组为对照组,RT-PCR法检测IFI16mRNA表达,流式细胞仪Annexin-V FITC/PI法检测细胞凋亡,Western blotting检测蛋白表达及细胞增殖信号通路相关蛋白磷酸化水平。结果:与阴性对照组比较,对照组及IFN-α组IFI16mRNA和蛋白表达上调,细胞凋亡增多,伴RAS蛋白表达减少,RAF、ERK磷酸化水平下降(P<0.01);IFN-α加IFI16siRNA组IFI16mRNA及蛋白表达下调,细胞凋亡减少,RAS蛋白表达增多,RAF及ERK磷酸化水平升高(P<0.01)。与对照组比较,IFN-α加IFI16siRNA组IFI16mRNA和蛋白表达减少,细胞凋亡减少,伴Ras蛋白表达增多,RAF、ERK磷酸化水平上调(P<0.01);而IFN-α组上述指标差异无统计学意义。在上述过程中,P38及AKT蛋白磷酸化水平无明显变化。结论:RAS信号途径参与IFI16siRNA抑制IFN-α诱导的HVECs凋亡。
AIM: To investigate the effect of interferon-inducible protein 16 (IFI16) siRNA on the apoptosis of human vascular endothelial cells (HVECs) induced by interferon-α (IFN-α) and its mechanism. Methods: The HVECs cultured in vitro were transiently transfected with IFN-α and / or IFI16 siRNA. The blank control group, IFN-α and non-specific siRNA transfected group were used as control group. The expression of IFI16 mRNA was detected by RT-PCR , Flow cytometry Annexin-V FITC / PI method to detect apoptosis, Western blotting protein expression and cell proliferation signaling pathway related protein phosphorylation. Results: Compared with the negative control group, the expression of IFI16 mRNA and protein in the control group and IFN-α group were upregulated, the apoptosis increased, the expression of RAS protein decreased, the phosphorylation of RAF and ERK decreased (P <0.01) Group IFI16mRNA and protein expression was downregulated, apoptosis decreased, RAS protein expression increased, RAF and ERK phosphorylation levels increased (P <0.01). Compared with the control group, IFI16 mRNA and protein expression decreased, apoptosis decreased, Ras protein expression increased, RAF and ERK phosphorylation levels increased in IFN-α plus IFI16siRNA group (P <0.01), while those in IFN-α group had no difference Statistical significance. In the above process, P38 and AKT protein phosphorylation levels did not change significantly. Conclusion: The involvement of RAS signaling pathway in IFI16 siRNA inhibits IFN-α-induced apoptosis of HVECs.