干扰素诱导蛋白204抑制大鼠血管外膜成纤维细胞增殖

Interferon-α Inducible Protein 204 Inhibition of Proliferation of Vascular Adventitial Fibroblasts in Rats

目的:探讨干扰素诱导蛋白204(p204)对大鼠血管外膜成纤维细胞(VAFs)增殖的影响及可能机制。方法:将大鼠VAFs分为空白对照组(N组)、非特异性小干扰RNA(siRNA)转染组(control组)、IFN-α干预组(IFN-α组)及Ifi204 siRNA转染组(Ifi204 siRNA组),给予相应处理;应用Real Time qRT-PCR法检测p204mRNA表达,MTT比色法测定细胞活力,流式细胞仪分析细胞周期,Western blot检测p204、Ras蛋白表达及Raf与Erk磷酸化水平。结果:与N组比较,IFN-α组p204 mRNA和蛋白表达上调(P<0.05),细胞活力下降,细胞周期G_1/S转换下调(P<0.05),伴Ras蛋白表达减少,Raf及Erk磷酸化水平下降(P<0.05);Ifi204 siRNA组p204 mRNA及蛋白表达下调(P<0.05),细胞活力增高,G_0/G_1期细胞减少,S期细胞增多(P<0.05),Ras蛋白表达增多,Raf及Erk磷酸化水平升高(P<0.05);而control组上述指标与N组相比,差异无统计学意义(P>0.05)。结论:p204表达可抑制大鼠VAFs增殖,Ras信号通路可能参与p204对VAFs增殖的调控。

Objective： To explore the effect of interferon inducible protein204（ p204） expression on rats＇ vascular adventitial fibroblasts（ VAF） proliferation and possible mechanism. Methods： The cultured VAFs were divided into four groups： blank comparison group（ N group）,nonspecific siRNA transfection group（ Control group）,IFN-α intervention group（ IFN-α group,treated with transfection of IFN-α） and Ifi204 siRNA transfection group（ Ifi204 siRNA group,treated with transfection of p204gene（ Ifi204） siRNA）. Expression of p204 mRNA was monitored by real time fluorescence quantitative reverse transcription polymerase chain reaction（ Real Time qRT-PCR）. Cell vitality was detected by 3-（ 4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（ MTT） method. Cell cycle was analyzed by flow cytometry. Proteins of 204 and Ras and the phosphorylation levels of Raf and Erk were examined by Western blot. Results： Compared with N group,in IFN-α intervention group,the expression of p204 mRNA and protein up-regulated（ P〈0. 05）,the cell vitality and the cell cycle of G_1/S transition down-regulated（ P〈0. 05）,the expression of Ras protein and the phosphorylation levels of Raf and Erk decreased（ P〈0. 05）; Compared with N group,in Ifi204 siRNA transfection group,the expression of p204 mRNA and protein down-regulated（ P〈0. 05）,the cell vitality increased,cells of G_0/ G_1 stage decreased and S stage increased（ P〈0. 05）,the expression of Ras protein and the phosphorylation levels of Raf and Erk increased（ P〈0. 05）; There were no statistically significant differences in above-mentioned indicators between Control group and N group（ P〈0. 05）. Conclusion： The expression of p204 can inhibit the proliferation of VAFs in rat,and Ras signaling pathway may participate in regulation of p204 on proliferation of VAFs.