线粒体融合素2基因突变体对大鼠血管平滑肌细胞增殖的影响

Effect of Mitofusin 2 Gene Mutations on the Proliferation of Vascular Smooth Muscle Cells

目的研究大鼠线粒体融合素2基因的蛋白激酶A磷酸化位点两种突变体对大鼠血管平滑肌细胞增殖的影响及其相关的信号通路。方法利用两种携带蛋白激酶A磷酸化位点突变的线粒体融合素2基因重组腺病毒(Adv-Mfn2-alaPKA和Adv-Mfn2-asnPKA)和携带线粒体融合素2基因的重组腺病毒(Adv-Mfn2),感染培养的大鼠血管平滑肌细胞。水溶性四甲基偶氮唑盐法比较各组细胞增殖的变化;流式细胞术比较各组细胞周期的变化;免疫印迹法比较各组线粒体融合素2基因和磷酸化细胞外调节激酶1/2蛋白表达变化。结果感染重组腺病毒的三组细胞线粒体融合素2基因表达差异无显著性。与对照组相比,Adv-Mfn2-alaPKA和Adv-Mfn2组显著抑制细胞增殖(P<0.01),停滞于G0/G1期细胞比例显著增加(P<0.01),磷酸化细胞外调节激酶1/2蛋白表达水平显著降低(P<0.01),且Adv-Mfn2-alaPKA作用更明显(P<0.01),而Adv-Mfn2-asnPKA组较对照组差异无显著性。结论Mfn2-alaPKA通过细胞外调节激酶1/2信号通路抑制大鼠血管平滑肌细胞增殖的作用较线粒体融合素2基因更明显;而Mfn2-asnPKA对血管平滑肌细胞无抑制增殖的作用,对细胞外调节激酶1/2信号通路也无作用。蛋白激酶A磷酸化位点是调控线粒体融合素2基因抗血管平滑肌细胞增殖的重要功能位点。

Aim To study the role of mutations of Mfn2 protein kinase A phosphorylation site in the proliferation of vascular smooth muscel cell（VSMC） and related signaling pathways. Methods Two novel mutations were constructed,Adv-Mfn2-αPKA and Adv-Mfn2-asnPKA,then VSMC were infected with them.The effect of mutations on the proliferation of VSMC was explored by WST-1 assay.The cell cycle was determined using flow cytometry.Western blotting were used to detect the expression of Mfn2 and p-ERK1/2. Results The expression of Mfn2 protein has no significant difference in Adv-Mfn2,Adv-Mfn2-alaPKA and Adv-Mfn2-asnPKA groups.Mfn2-alaPKA and Mfn2 both showed stronger inhibitory effect on VSMC proliferation（P〈0.01）.Most of the cells in these two groups were blocked in the stage of G0/G1（P〈0.01）,and the expression levels of p-ERK1/2 also decrease（P〈0.01）.Mfn2-alaPKA is superior to Mfn2 in attenuating the proliferation of VSMCs.The effect of Mfn2-asnPKA is similar to the control. Conclusion Mfn2-ala PKA has more stronger inhibitory effect on the proliferation of VSMC than Mfn2,while Mfn2-asnPKA has no effect.PKA phosphorylation site plays an important role in regulating the function of Mfn2 gene.