【作者】 周炜；

【导师】 郭小梅；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 第一部分去除PKA磷酸化位点对Mfn2基因调控细胞增殖和凋亡功能的影响1.目的研究大鼠线粒体融合素基因2(Mitofusin 2, Mfn2)在去除蛋白激酶A (Protein kinaseA, PKA)磷酸化位点后对大鼠血管平滑肌细胞(Vascular smooth muscle cells, VSMCs)增殖和凋亡的影响及相关的信号通路。2.方法利用携带去除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)感染大鼠VSMCs。激光共聚焦显微镜观察其细胞内定位；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；细胞凋亡ELISA分析其对细胞凋亡的影响；Western blot法分析Mfn2-PKA(△)、Mfn2、磷酸化ERK1／2(p-ERKl／2)和磷酸化Akt(p-Akt)蛋白的表达变化。3.结果外源基因转染后表达特异性蛋白产物；Mfn2-PKA(△)主要分布于线粒体上,与Mfn2相同；Mfn2-PKA(△)抑制VSMCs增殖、诱导VSMCs凋亡的作用较Mfn2显著减弱(P<0.01),与对照组无显著差异；Mfn2-PKA(△)较Mfn2组p-ERK1／2表达显著升高(P<0.01),p-Akt表达也显著升高(P<0.01),与对照组无明显差异。4.结论去除PKA磷酸化位点不影响Mfn2在线粒体上的定位,但其调节VSMCs增殖和凋亡的作用消失,对ERK1／2和Akt信号通路也无抑制作用。表明PKA磷酸化位点对Mfn2调节VSMCs增殖和凋亡的功能有重要影响。第二部分Mfn2磷酸化位点突变体抑制细胞增殖作用的差异1.目的研究大鼠线粒体融合素2基因(Mitofusin2, Mfn2)蛋白激酶A (Protein kinase A,PKA)磷酸化位点的状态对大鼠VSMCs增殖的影响及其相关的信号通路。2.方法利用携带PKA磷酸化位点突变的Mfn2重组腺病毒(Adv-Mfn2-S442A和Adv-Mfn2-S442D)和携带Mfn2的重组腺病毒(Adv-Mfn2),感染大鼠VSMCs。激光共聚焦法分析Mfn2及其突变体对线粒体形态的影响。细胞计数法、水溶性四甲基偶氮唑盐(WST-1)法比较其对细胞增殖的影响；流式细胞术比较各组细胞周期的变化；Western blot法分析各组Mfn2、磷酸化Raf-1(p-Raf-1)、磷酸化ERK1／2(p-ERKl／2)蛋白表达变化。建立大鼠颈动脉球囊损伤再狭窄模型,局部血管转染Mfn2及突变体基因,HE染色观察颈动脉内膜增殖程度,免疫组化染色观察增殖细胞核抗原(PCNA)的表达水平。3.结果外源基因转染后表达特异性蛋白产物,Mfn2及其突变体均促进线粒体融合并聚集于核周。细胞实验,Adv-Mfn2-S442A和Adv-Mfn2抑制细胞增殖作用较对照组显著增强(P<0.01),停滞于G0／G1期细胞比例显著增加(P<0.01)；p-Raf-1和p-ERK1／2表达水平显著降低(P<0.01),且Adv-Mfn2-S442A作用更明显(P<0.01),而Adv-Mfn2-S442D组较对照组无显著差异。动物实验,Adv-Mfn2-S442A和Adv-Mfn2组大鼠颈动脉球囊损伤后内膜增殖较对照组显著减弱(P<0.01),PCNA表达水平显著减弱(P<0.01),且Adv-Mfn2-S442A作用更明显(P<0.01),而Adv-Mfn2-S442D组较对照组无显著差异。4.结论去磷酸化的Mfn2通过ERK1／2信号通路抑制VSMCs增殖的作用较Mfn2更明显；而磷酸化的Mfn2无显著作用。PKA磷酸化位点的状态对Mfn2调节VSMCs增殖有重要影响,并独立于Mfn2促进线粒体融合的作用。第三部分Mfn2磷酸化位点突变体诱导细胞凋亡作用的差异1.目的研究大鼠线粒体融合素2基因(Mitofusin2, Mfn2)蛋白激酶A (Protein kinase A,PKA)磷酸化位点的状态对大鼠VSMCs凋亡的影响及其相关的信号通路。2.方法利用携带PKA磷酸化位点突变的Mfn2重组腺病毒(Adv-Mfn2-S442A和Adv-Mfn2-S442D)和携带Mfn2的重组腺病毒(Adv-Mfn2),感染大鼠VSMCs。流式细胞术比较各组细胞凋亡率的变化；JC-1染色法检测线粒体膜电位变化；Westernblot法分析各组Mfn2、磷酸化Akt(p-Akt)和活性半胱天冬酶9(cleaved caspase-9)蛋白表达变化。3.结果外源基因转染后表达特异性蛋白产物。Adv-Mfn2-S442A和Adv-Mfn2诱导细胞凋亡的作用较对照组显著增强(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),cleaved caspase-9表达水平显著增高(P<0.01),且Adv-Mfn2-S442A作用更明显(P<0.01),而Adv-Mfn2-S442D组较对照组无显著差异。4.结论去磷酸化的Mfn2通过Akt信号通路诱导VSMCs凋亡的作用较Mfn2更明显；而磷酸化的Mfn2无显著作用,PKA磷酸化位点的状态对Mfn2调节VSMCs凋亡有重要影响。更多还原

【Abstract】 PartⅠEffect of mitofusin 2 with protein kinase A phosphorylation site deletion on the proliferation and apoptosis of vascular smooth muscle cells1. ObjectiveTo investigate the effect of mitofusin 2 (Mfn2) gene with the protein kinase A (PKA) phosphorylation site deletion [Mfn2-PKA(△)] on inhibiting the proliferation and inducing the apoptosis of vascular smooth muscle cells (VSMCs) and related signaling pathway.2. MethodsVSMCs of rats were infected by recombinational adenovirus carrying green fluorescent protein (GFP), Mfn2-PKA (△) or Mfn2 gene (Adv-GFP, Adv-Mfn2-PKA (△), Adv-Mfn2). The abundance of Mfn2-PKA(△) protein and Mfn2 protein were determined by Western blot analysis using Mfn2 antibody. Laser confocal microscopy (LCMS) was used to observe the locations of the proteins. The growth curve of the VSMCs was explored by MTT. The effect of Adv-Mfn2-PKA(△) on the apoptosis of VSMCs was explored by ELISA. Western blot were used to detect the expression of phosphorylation of ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt).3. ResultsThe Mfn2 and Mfn2-PKA (△) both expressed protein-specific bands in VSMCs. Two kinds of gene expression products were mainly located at the out membrane of mitochondria. Compared with the control group and Adv-GFP group, the absorbance values at 3,4,5,6 days were significantly reduced in Adv-Mfn2 group (P< 0.01), and no obvious changes were observed in Adv-Mfn2-PKA (△) group. Mfn2-PKA(△) had no effect on promoting the apoptosis of VSMCs compared with Mfn2 (P<0.01). Overexpression of Mfn2-PKA(△) gene could not down-regulate the expression of p-ERK1/2 and p-Akt (P< 0.01).4. ConclusionsMfn2-PKA (△), located at the out membrane of mitochondria, has no effect on suppressing the proliferation and on inducing apoptosis of VSMCs. Mfn2-PKA (△) has no inhibition effect on ERK1/2 and Akt signaling pathway. PKA phosphorylation site plays an important role in regulating the function of Mfn2 gene.PartⅡMutation of the protein kinase A phosphorylation site influences the anti-proliferative activity of mitofusin 21. ObjectiveMitofusin 2 (Mfn2) is an important suppressor of vascular smooth muscle cells (VSMCs) proliferation. It contains a protein kinase A (PKA) phosphorylation site at serine 442 (S442) and can be phosphorylated by PKA. This study examined the role of phosphorylating specific sites on the regulation of Mfn2 protein activity in vitro and in vivo.2. MethodsTo investigate the functional significance of Mfn2 phosphorylation, we introduced two separate mutations at the codon for serine 442 in the rat Mfn2 gene. Replacing serine with alanine provided a nonphosphorylatable residue with minimal structural changes to the protein, while replacing the serine residue with aspartic acid mimicked constitutive phosphorylation at this site. In this study, we evaluated changes in Mfn2 protein abundance, mitochondrial morphology and membrane fusion in adenovirus infected rat VSMCs. Since Mfn2 has profound suppressive effects on Ras in VSMCs and might therefore inhibit cell proliferation by modulating the activity of the Ras-Raf-ERK1/2 signaling pathway and the cell cycle, we chose to test the effects of these mutations on proliferation of VSMCs in vitro and in vivo.3. ResultsOur results indicated that, in VSMCs, Mfn2 expression and mitochondrial morphology are affected by adenoviral-mediated overexpression of the two Mfn2 mutant proteins in the same way as the wild-type Mfn2 protein. Specifically, overexpression of the protein harboring the phospho-deficient mutation Mfn2-S442A (serine replaced by alanine at residue 442) increased the inhibitory effects of Mfn2 on proliferation of VSMCs, and stimulation effects on apoptosis of VSMCs in culture. On the other hand, the phospho-mimetic mutation Mfn2-S442D (serine replaced by aspartic acid at residue 442) led to loss of growth suppressor activity.4. ConclusionsThese results suggest that this specific PKA phosphorylation site plays a key role in Mfn2-mediated suppression of VSMC growth, which is independent of its effects on modulation of mitochondrial morphology.PartⅢMutation of the protein kinase A phosphorylation site influences the pro-apoptosis activity of mitofusin 21. ObjectiveTo study the role of Mfn2 gene with protein kinase A phosphorylation site mutations in the apoptosis of VSMCs and related signaling pathways. 2. MethodsTwo novel mutations were constructed, Adv-Mfn2-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 using JC-1 staining method. Western blot were used to detect the expression of Mfn2、p-Akt and cleaved caspase-9.3. ResultsThe expression of Mfn2 protein has no significant difference in Adv-Mfn2, Adv-Mfn2-S442A and Adv-Mfn2-S442D groups. Flow cytometry analysis showed both of Mfn2-S442A and Mfn2 had stronger effect in promoting the apoptosis of VSMCs (P<0.01). The mitochondrial membrane potential in these two groups remarkably decreased (P<0.01). The results of Western blot indicated that the protein expression of p-Akt remarkably decreased, whereas cleaved capased-9 protein highly expressed in Adv-Mfn2-S442A and Adv-Mfn2 group (P<0.01). Mfn2-S442A was superior to Mfn2 in promoting the apoptosis of VSMCs (P<0.01). The effect of Mfn2-S442D was similar to the control.4. ConclusionsMfn2-S442A has more stronger inducing effect on the apoptosis of VSMCs than Mfn2, while Mfn2-S442D has no effect. We can conclude that PKA phosphorylation site plays an important role in regulating the function of Mfn2 gene.更多还原