tMfn2基因抑制血管平滑肌细胞增殖的作用与机制

Adenovirus mediated tMfn2 gene inhibited the proliferation of vascular smooth muscle cells

目的比较线粒体融合素基因2（Mfn2）和去除穿膜区序列的大鼠线粒体融合素基因2（tMfn2）对大鼠血管平滑肌细胞（VSMCs）增殖的作用，探讨tMfn2基因对VSMCs增殖的影响及其相关的信号通路。方法用携带tMfn2基因和Mfn2基因的重组腺病毒（Adv—tMfn2和Adv-Mfn2）感染VSMCs，检测tMfn2蛋白和Mfn2蛋白在细胞中的表达水平；细胞计数法、四甲基偶氮唑盐（MTT）法检测VSMCs的增殖；流式细胞术检测tMfn2基因对VSMCs周期的影响；Western blot分析各组磷酸化ERK1／2和磷酸化Raf-1蛋白表达水平的变化；采用方差分析对数据进行统计学处理。结果Adv-tMfn2和Adv—Mfn2感染VSMCs后能有效表达出相应蛋白；细胞计数和MTT结果显示，tMfn2和Mfn2均使VSMCs增殖受到明显抑制（P〈0．01），且前者较后者抑制效果更明显（P〈0．01）；流式细胞术结果表明tMfn2和Mfn2使多数VSMCs停滞于G0／G1期，细胞比例为（88．01±4．38）％和（67．43±6．21）％，可见tMfn2基因对细胞周期的影响更明显（P〈0．01）。进一步研究表明tMfn2比Mfn2更能降低磷酸化ERK1／2（p-ERK1／2）和磷酸化Raf-1（p-Raf-1）的表达水平（P〈0．01）。结论与Mfn2基因相比，tMfn2基因更能显著抑制VSMCs增殖，使细胞周期停滞于G0／G1期。这一作用主要通过抑制Ras-Raf-ERK1／2信号通路，下调磷酸化Raf-1蛋白表达，进而抑制ERK1／2的磷酸化而实现。

Objective To investigate the effect of tMfn2 gene on inhibiting the proliferation of vascular smooth muscle cells （VSMCs） and related mechanism. Method VSMCs were transfected with adenovirus vector encoding tMfn2 or Mfn2 （Adv-tMfn2, Adv-Mfn2）. The abundance of tMfn2 protein and Mfn2 protein were determined by Western blot analysis using Mfn2 N-term antibody. The effect of tMfn2 on the proliferation of VSMCs was explored by cell counting and MTT assay. The cell cycle was analyzed using flow cytometry. Western blot were used to detect the expression of p-ERK1/2 and p-Raf-l. Results The results of cell counting and MTF both indicated that tMfn2 gene displayed more remarkable effect on inhibiting the proliferation of VSMCs than Mfn2 （ P 〈 0. 01）. How cytometry showed that most of the cells infected with Adv-tMfn2 or Adv-Mfn2 were blocked in the stage of G0/G1 and few entered into the S phase. Western blot indicated that overexpression of tMfn2 gene resulted in dowrtregulation of phosphorylated ERK1/2 and Raf-1 protein （ P 〈 0.01 ）. These results demonstrated tMfn2 had stronger effect than Mfn2 （ P 〈 0.01 ）. Conclusions tMfn2 gene is superior to Mfn2 gene in attenuating the proliferation of VSMCs via the Ras-Raf-ERK1/2 signaling pathway.