Mfn2基因PKA磷酸化位点的功能研究

发布时间：2018-05-02 21:19

  本文选题：mitofusin + 2　； 参考：《华中科技大学》2010年博士论文

【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(P0.01),与对照组无显著差异；Mfn2-PKA(△)较Mfn2组p-ERK1／2表达显著升高(P0.01),p-Akt表达也显著升高(P0.01),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(P0.01),停滞于G0／G1期细胞比例显著增加(P0.01)；p-Raf-1和p-ERK1／2表达水平显著降低(P0.01),且Adv-Mfn2-S442A作用更明显(P0.01),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(P0.01),PCNA表达水平显著减弱(P0.01),且Adv-Mfn2-S442A作用更明显(P0.01),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(P0.01),线粒体膜电位显著降低(P0.01),p-Akt表达水平显著降低(P0.01),cleaved caspase-9表达水平显著增高(P0.01),且Adv-Mfn2-S442A作用更明显(P0.01),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。
[Abstract]:Part one
Effect of removing PKA phosphorylation sites on Mfn2 gene regulation of cell proliferation and apoptosis
1. purposes
The effects of mitochondrial fusion gene 2 (Mitofusin 2, Mfn2) on the proliferation and apoptosis of rat vascular smooth muscle cells (Vascular smooth muscle cells, VSMCs) and the related signaling pathways were investigated after the phosphorylation sites of protein kinase A (Protein kinaseA, PKA) were removed.
2. method
The intracellular localization of the Mfn2 recombinant adenovirus carrying the recombinant adenovirus carrying PKA phosphorylation site and the recombinant adenovirus carrying Mfn2 (Adv-Mfn2) infected rat VSMCs. laser confocal microscope was observed. The morphological changes of the cells were observed by the fluorescence microscope, and the effect of four methyl azazolium salt (MTT) on the cell proliferation was compared. The apoptosis was analyzed by ELISA, and the expression of Mfn2-PKA (delta), Mfn2, phosphorylated ERK1 / 2 (p-ERKl / 2) and phosphorylated Akt (p-Akt) protein were analyzed by Western blot method.
3. results
The specific protein products were expressed by exogenous gene transfection; Mfn2-PKA (delta) was mainly distributed on the mitochondria, the same as that of the Mfn2. Mfn2-PKA (delta) inhibited the proliferation of VSMCs and induced the apoptosis of VSMCs significantly than Mfn2 (P0.01), no significant difference from the control group; Mfn2-PKA (delta) was significantly higher than the Mfn2 group p-ERK1 / 2 (P0.01), and p-Akt expression also showed There was no significant difference between the control group and the increase (P0.01).
4. conclusion
The removal of PKA phosphorylation site does not affect the localization of Mfn2 on the mitochondria, but its role in regulating the proliferation and apoptosis of VSMCs disappears, and has no inhibitory effect on the ERK1 / 2 and Akt signaling pathways. It is indicated that the PKA phosphorylation site has an important effect on the function of Mfn2 to regulate the proliferation and apoptosis of VSMCs.
The second part
Differences in inhibitory effect of Mfn2 phosphorylation site mutants on cell proliferation
1. purposes
To study the effect of the state of phosphorylation of Mitofusin2 (Mitofusin2, Mfn2) protein kinase A (Protein kinase A, PKA) on the proliferation of rat VSMCs and its related signaling pathway.
2. method
Mfn2 recombinant adenovirus (Adv-Mfn2-S442A and Adv-Mfn2-S442D) and recombinant adenovirus carrying Mfn2 (Adv-Mfn2) were used to carry the mutation of PKA phosphorylation site. The effect of Mfn2 and its mutants on mitochondrial morphology was analyzed by VSMCs. laser confocal method in rats. Cell count method, water-soluble four methylazazolium salt (WST-1) method compared its cell growth. Effect of colonization; flow cytometry was used to compare the changes of cell cycle in each group; Western blot method was used to analyze the changes of Mfn2, phosphorylated Raf-1 (p-Raf-1), phosphorylated ERK1 / 2 (p-ERKl / 2) protein expression. The rat carotid balloon injury restenosis model was established, local blood vessels were transfected with Mfn2 and mutant genes, and the intima proliferation of carotid artery was observed by HE staining. Immunohistochemical staining was used to observe the expression level of proliferating cell nuclear antigen (PCNA).
3. results
The expression of specific protein products was expressed by exogenous gene transfection. Both Mfn2 and its mutants promoted mitochondrial fusion and aggregated in the perinuclear cycle. Cell proliferation was significantly enhanced by Adv-Mfn2-S442A and Adv-Mfn2 (P0.01), and the percentage of stagnation in G0 / G1 phase was significantly increased (P0.01), p-Raf-1 and p-ERK1 / 2 expression levels were significantly reduced. Low (P0.01), and the effect of Adv-Mfn2-S442A was more obvious (P0.01), but there was no significant difference between the group Adv-Mfn2-S442D and the control group. In the animal experiment, the intimal proliferation of the carotid balloon injury in the Adv-Mfn2-S442A and Adv-Mfn2 rats was significantly weakened (P0.01), the expression of PCNA was decreased (P0.01), and the Adv-Mfn2-S442A effect was more obvious (P0.01), and Adv-Mf was Adv-Mf. There was no significant difference between the n2-S442D group and the control group.
4. conclusion
The effect of dephosphorylation of Mfn2 through the ERK1 / 2 signaling pathway to inhibit VSMCs proliferation is more obvious than that of Mfn2, while phosphorylated Mfn2 has no significant effect on.PKA phosphorylation sites and has an important effect on Mfn2 regulation of VSMCs proliferation, and is independent of Mfn2 to promote mitochondrial fusion.
The third part
Differences in apoptosis induced by Mfn2 phosphorylation site mutants
1. purposes
To study the effect of the state of phosphorylation of Mitofusin2 (Mitofusin2, Mfn2) protein kinase A (Protein kinase A, PKA) on the apoptosis of rat VSMCs and its related signaling pathway.
2. method
The Mfn2 recombinant adenovirus (Adv-Mfn2-S442A and Adv-Mfn2-S442D) and the recombinant adenovirus carrying Mfn2 (Adv-Mfn2) with the mutation of the PKA phosphorylation site were used to infect the rat VSMCs. flow cytometry to compare the changes in the apoptosis rate of each group; JC-1 staining method was used to detect the mitochondrial membrane potential change; Westernblot method was used to analyze Mfn2, Akt phosphorylation (p-Ak). T and the expression of active caspase 9 (cleaved caspase-9) protein.
3. results
The expression of specific protein products.Adv-Mfn2-S442A and Adv-Mfn2 induced apoptosis significantly increased (P0.01), mitochondrial membrane potential decreased significantly (P0.01), p-Akt expression level decreased significantly (P0.01), cleaved caspase-9 expression level increased significantly (P0.01), and Adv-Mfn2-S442A effect was more obvious (P0.01). There was no significant difference between the Adv-Mfn2-S442D group and the control group.
4. conclusion
The effect of dephosphorylation of Mfn2 through Akt signaling pathway to induce VSMCs apoptosis is more obvious than that of Mfn2, while phosphorylation Mfn2 has no significant effect. The state of phosphorylation site of PKA has an important effect on Mfn2 regulation of VSMCs apoptosis.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R341

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