线粒体融合素2突变体对血管平滑肌细胞凋亡的影响  

Effect of mitofusin 2 mutations on the apoptosis of vascular smooth muscle cells

作　　者：周炜[1] 曹文静[1] 陈莉莉[1] 郭小梅[1] 陈光慧[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院心内科,武汉430030 [2]美国国立卫生研究院

出　　处：《中华老年心脑血管病杂志》2010年第2期154-157,共4页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：国家自然科学基金(30672206;30600233)

摘　　要：目的研究大鼠线粒体融合素2(mitofusin 2,Mfn2)基因蛋白激酶A(PKA)磷酸化位点的2种突变体对大鼠血管平滑肌细胞(VSMCs)凋亡及其相关的信号通路的影响。方法构建4种重组腺病毒,分别携带磷酸化位点突变为丙氨酸(重组1组)、Mfn2基因(重组2组)、突变为天冬酰胺(重组3组)和半乳糖苷酶基因(对照组),感染培养的VSMCs,另设未感染腺病毒的空白组。流式细胞术比较各组细胞的凋亡率,JC-1染色法检测线粒体膜电位变化,Western blot分析各组Mfn2蛋白的表达以及磷酸化蛋白激酶B(p-Akt)和活性半胱天冬酶9(caspase-9)表达。结果与空白组和对照组比较,重组1组、重组2组和重组3组Mfn蛋白显著增加(P<0.01);重组1组和重组2组细胞凋亡率显著增强(P<0.01);线粒体膜电位显著降低(P<0.01);p-Akt表达水平显著降低(P<0.01),活性caspase-9表达水平显著增高(P<0.01);且重组1组作用较重组2组更明显(P<0.01);而重组3组上述指标无显著差异(P>0.05)。结论 Mfn2突变为丙氨酸的位点PKA通过Akt信号及线粒体途径诱导VSMCs凋亡的作用较Mfn2更明显,表明PKA磷酸化位点是调控Mfn2诱导VSMCs凋亡的重要功能位点。Objective To study the effect of mitofusin 2（Mfn2） gene with protein kinase A（PKA） phosphorylation site mutations on the apoptosis of VSMCs and related signaling pathways. Methods Four recombinant adenoviruses carting two novel mutations of Mfn2 were constructed,AdvMfn2-alaPKA and Adv-Mfn2-asnPKA, then VSMCs were infected with them. The effect of mutations on the apoptosis of VSMCs was explored by flow cytometry analysis. The cell mitochondrial membrane potential was detected by JC-1 staining method. Western blot was used to detect the expression of Mfn2,p-Akt and cleaved caspase-9. Results The expression of Mfn2 protein had no significant difference among Adv-Mfn2,Adv-Mfn2-alaPKA and Adv-Mfn2-asnPKA groups. Compared with control group,both Mfn2-alaPKA and Mfn2 had stronger effect in promoting the apoptosis of VSMCs （P 〈 0.01）. In these 2 groups, the mitochondrial membrane potential decreased （P % 0.01）, the protein expression of p-Akt remarkably decreased, whereas cleaved capase-9 protein expression increased. Mfn2-alaPKA was superior to Mfn2 in promoting the apoptosis of VSMCs. Conclusions Mfn2-alaPKA has stronger promoting effect on the apoptosis of VSMCs than Mfn2,while Mfn2-asnPKA has no effect. PKA phosphorylation site plays an important role in regulating the function of Mfn2 gene.

关 键 词：线粒体 点突变 肌 平滑 血管 细胞凋亡 蛋白激酶类 

分 类 号：R363[医药卫生—病理学]