线粒体融合素基因2突变体Mfn2-1A抑制大鼠血管平滑肌细胞增殖及机制研究

Mitochondrial fusion gene 2 mutant inhibits the proliferation of vascular smooth muscle cells and its mechanism

目的:研究线粒体融合素基因2(Mfn2)的突变体片段Mfn2-1A,对自发性高血压大鼠血管平滑肌细胞的增殖抑制作用,并进一步探讨相关作用机制。方法:用已构建的重组腺病毒Adv-Mfn2-1A感染大鼠血管平滑肌细胞,利用细胞计数、四甲基偶氮唑盐比色法检测血管平滑肌细胞增殖,流式细胞仪检测细胞生长周期,蛋白免疫印迹法分析Mfn2-1A对信号通路Mek-Erk1/2蛋白表达水平的影响。结果:重组腺病毒Adv-Mfn2-1A感染平滑肌细胞能正常表达相应蛋白;转染Adv-Mfn2-1A后能抑制平滑肌细胞增殖(P<0.05),第3天开始效果明显;Adv-Mfn2-1A较Adv-Mfn2作用效果更显著(P<0.05);转染Adv-Mfn2-1A和Adv-Mfn2后阻滞于G0/G1期的细胞明显增多,转染Mfn2-1A后阻滞在G0/G1期细胞达到(77.74±3.67)%;Mfn2-1A可降低平滑肌细胞中磷酸化Erk1/2蛋白的表达,效果比Mfn2更明显(P<0.05)。结论:Mfn2基因突变体片段1A可明显抑制血管平滑肌增殖,使细胞阻滞在G0/G1期,其机制与抑制Mek-Erk1/2信号通路有关。

Objective：To investigate the effect of Mfn21A on the proliferation of VSMC and its underlying mechanism. Method：The fragment sequence of Mfn21A contains the p21 ras signature motif and ATP/GTP binding site motif.VSMCs separated from spontaneously hypertensive rats were transfected with adenovirus mediated 1A（Adv-Mfn2 1A） or adenovirus mediated Mfn2（Adv-Mfn2）.The proliferation of VSMC was detemined by cell counting and CCK-8.The cell cycle was analyzed by flow cytometry.The protein expression of Mek and Erk1/2 was determined by Western blot. Result：VSMCs tranfected with Adv-Mfn2 1A could express Mfn2 protein.Overexpression of Mfn21A inhibited the proliferation of VSMC more obviously than Mfn2 on day 3（P〈0.05）.More cells were arrested at G0/G1 phases after Mfn2 1A and Mfn2 overexpression,and the percentage of cell that were arrested at G0/G1 phases was（77.74±3.67）% in Mfn21A transfection group.Overexpression of Mfn2 1A led to down regulation of P-Erk1/2,and this downregulation was more significant in Mfn21A transfection group than that of Mfn2 group（P〈0.05）. Conclusion：Overexpression of Mfn2 1A inhibit the proliferation of on VSMC and induce cell cycle arrest at G0/G1 phases via inhibiting the Mek-Erk1/2 signaling pathway.