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目的:研究大鼠线粒体融合素基因2(Mfn2)在去除蛋白激酶A(PKA)磷酸化位点后对大鼠血管平滑肌细胞(VSMC)增殖的影响及其相关的信号通路。方法:构建携带去除PKA磷酸化位点的Mfn2重组腺病毒[Adv-Mfn2-PKA(△)]和携带Mfn2的重组腺病毒(Adv-Mfn2)并感染VSMC。Western blot分析法Mfn2-PKA(△)和Mfn2蛋白的表达;激光共聚焦显微镜观察其亚细胞定位;荧光显微镜观察细胞形态变化;四甲基偶氮唑盐(MTT)法比较其对细胞增殖的影响;Western blot法分析磷酸化ERK1/2(p-ERK1/2)蛋白表达变化。结果:激光共聚焦显微镜示Mfn2-PKA(△)与Mfn2蛋白都主要分布于线粒体外膜;荧光显微镜示Mfn2组细胞数较少,而Mfn2-PKA(△)组与对照组相似;MTT示,Mfn2-PKA(△)抑制VSMC增殖作用较Mfn2显著减弱(P<0.01),与对照组无显著差异;Westernblot结果显示,Mfn2-PKA(△)较Mfn2组p-ERK1/2表达显著升高(P<0.01),与对照组无明显差异。结论:Mfn2-PKA(△)与Mfn2蛋白一样都定位于线粒体外膜,但抑制VSMC增殖的作用消失,对ERK1/2信号通路无抑制作用。表明PKA磷酸化位点是调控Mfn2抗VSMC增殖的重要功能位点。
AIM: To investigate the effects of Mfn2 on the proliferation of rat vascular smooth muscle cells (VSMCs) after its excision of PKA phosphorylation sites and its related signaling pathways. Methods: Mfn2 recombinant adenovirus [Adv-Mfn2-PKA (Δ)] carrying Mfn2 and adenovirus carrying Mfn2 (Adv-Mfn2) carrying PKA phosphorylation site were constructed and infected into VSMC. The expression of Mfn2-PKA (△) and Mfn2 protein was detected by Western blotting. The subcellular localization was observed by laser confocal microscopy. The morphological changes of cells were observed by fluorescence microscopy. The MTT assay was used to compare the expression of Mfn2- Western blot analysis of phosphorylated ERK1 / 2 (p-ERK1 / 2) protein expression changes. Results: The Mfn2-PKA (△) and Mfn2 proteins were mainly distributed in the mitochondrial outer membrane by laser scanning confocal microscopy; Mfn2-PKA (△) group was less than that of the control group by fluorescence microscopy; Mfn2-PKA (△) inhibited the proliferation of VSMC significantly (P <0.01), and had no significant difference with that of control group. Western blot showed that the expression of p-ERK1 / 2 was significantly increased in Mfn2-PKA P <0.01), no significant difference with the control group. CONCLUSION: Mfn2-PKA (△) locates on the mitochondrial outer membrane as well as the Mfn2 protein, but the effect of suppressing the proliferation of VSMC disappears and does not inhibit the ERK1 / 2 signaling pathway. It is indicated that PKA phosphorylation sites are important functional sites that regulate the proliferation of Mfn2 against VSMC.