本文選題：mitofusin + 2　； 參考：《華中科技大學(xué)》2010年博士論文

【摘要】： 第一部分 去除PKA磷酸化位點(diǎn)對(duì)Mfn2基因調(diào)控細(xì)胞增殖和凋亡功能的影響 1.目的 研究大鼠線粒體融合素基因2(Mitofusin 2, Mfn2)在去除蛋白激酶A (Protein kinaseA, PKA)磷酸化位點(diǎn)后對(duì)大鼠血管平滑肌細(xì)胞(Vascular smooth muscle cells, VSMCs)增殖和凋亡的影響及相關(guān)的信號(hào)通路。 2.方法 利用攜帶去除PKA磷酸化位點(diǎn)的Mfn2重組腺病毒[Adv-Mfn2-PKA(△)]和攜帶Mfn2的重組腺病毒(Adv-Mfn2)感染大鼠VSMCs。激光共聚焦顯微鏡觀察其細(xì)胞內(nèi)定位；熒光顯微鏡觀察細(xì)胞形態(tài)變化；四甲基偶氮唑鹽(MTT)法比較其對(duì)細(xì)胞增殖的影響；細(xì)胞凋亡ELISA分析其對(duì)細(xì)胞凋亡的影響；Western blot法分析Mfn2-PKA(△)、Mfn2、磷酸化ERK1／2(p-ERKl／2)和磷酸化Akt(p-Akt)蛋白的表達(dá)變化。 3.結(jié)果 外源基因轉(zhuǎn)染后表達(dá)特異性蛋白產(chǎn)物；Mfn2-PKA(△)主要分布于線粒體上,與Mfn2相同；Mfn2-PKA(△)抑制VSMCs增殖、誘導(dǎo)VSMCs凋亡的作用較Mfn2顯著減弱(P0.01),與對(duì)照組無(wú)顯著差異；Mfn2-PKA(△)較Mfn2組p-ERK1／2表達(dá)顯著升高(P0.01),p-Akt表達(dá)也顯著升高(P0.01),與對(duì)照組無(wú)明顯差異。 4.結(jié)論 去除PKA磷酸化位點(diǎn)不影響Mfn2在線粒體上的定位,但其調(diào)節(jié)VSMCs增殖和凋亡的作用消失,對(duì)ERK1／2和Akt信號(hào)通路也無(wú)抑制作用。表明PKA磷酸化位點(diǎn)對(duì)Mfn2調(diào)節(jié)VSMCs增殖和凋亡的功能有重要影響。 第二部分 Mfn2磷酸化位點(diǎn)突變體抑制細(xì)胞增殖作用的差異 1.目的 研究大鼠線粒體融合素2基因(Mitofusin2, Mfn2)蛋白激酶A (Protein kinase A,PKA)磷酸化位點(diǎn)的狀態(tài)對(duì)大鼠VSMCs增殖的影響及其相關(guān)的信號(hào)通路。 2.方法 利用攜帶PKA磷酸化位點(diǎn)突變的Mfn2重組腺病毒(Adv-Mfn2-S442A和Adv-Mfn2-S442D)和攜帶Mfn2的重組腺病毒(Adv-Mfn2),感染大鼠VSMCs。激光共聚焦法分析Mfn2及其突變體對(duì)線粒體形態(tài)的影響。細(xì)胞計(jì)數(shù)法、水溶性四甲基偶氮唑鹽(WST-1)法比較其對(duì)細(xì)胞增殖的影響；流式細(xì)胞術(shù)比較各組細(xì)胞周期的變化；Western blot法分析各組Mfn2、磷酸化Raf-1(p-Raf-1)、磷酸化ERK1／2(p-ERKl／2)蛋白表達(dá)變化。建立大鼠頸動(dòng)脈球囊損傷再狹窄模型,局部血管轉(zhuǎn)染Mfn2及突變體基因,HE染色觀察頸動(dòng)脈內(nèi)膜增殖程度,免疫組化染色觀察增殖細(xì)胞核抗原(PCNA)的表達(dá)水平。 3.結(jié)果 外源基因轉(zhuǎn)染后表達(dá)特異性蛋白產(chǎn)物,Mfn2及其突變體均促進(jìn)線粒體融合并聚集于核周。細(xì)胞實(shí)驗(yàn),Adv-Mfn2-S442A和Adv-Mfn2抑制細(xì)胞增殖作用較對(duì)照組顯著增強(qiáng)(P0.01),停滯于G0／G1期細(xì)胞比例顯著增加(P0.01)；p-Raf-1和p-ERK1／2表達(dá)水平顯著降低(P0.01),且Adv-Mfn2-S442A作用更明顯(P0.01),而Adv-Mfn2-S442D組較對(duì)照組無(wú)顯著差異。動(dòng)物實(shí)驗(yàn),Adv-Mfn2-S442A和Adv-Mfn2組大鼠頸動(dòng)脈球囊損傷后內(nèi)膜增殖較對(duì)照組顯著減弱(P0.01),PCNA表達(dá)水平顯著減弱(P0.01),且Adv-Mfn2-S442A作用更明顯(P0.01),而Adv-Mfn2-S442D組較對(duì)照組無(wú)顯著差異。 4.結(jié)論 去磷酸化的Mfn2通過ERK1／2信號(hào)通路抑制VSMCs增殖的作用較Mfn2更明顯；而磷酸化的Mfn2無(wú)顯著作用。PKA磷酸化位點(diǎn)的狀態(tài)對(duì)Mfn2調(diào)節(jié)VSMCs增殖有重要影響,并獨(dú)立于Mfn2促進(jìn)線粒體融合的作用。 第三部分 Mfn2磷酸化位點(diǎn)突變體誘導(dǎo)細(xì)胞凋亡作用的差異 1.目的 研究大鼠線粒體融合素2基因(Mitofusin2, Mfn2)蛋白激酶A (Protein kinase A,PKA)磷酸化位點(diǎn)的狀態(tài)對(duì)大鼠VSMCs凋亡的影響及其相關(guān)的信號(hào)通路。 2.方法 利用攜帶PKA磷酸化位點(diǎn)突變的Mfn2重組腺病毒(Adv-Mfn2-S442A和Adv-Mfn2-S442D)和攜帶Mfn2的重組腺病毒(Adv-Mfn2),感染大鼠VSMCs。流式細(xì)胞術(shù)比較各組細(xì)胞凋亡率的變化；JC-1染色法檢測(cè)線粒體膜電位變化；Westernblot法分析各組Mfn2、磷酸化Akt(p-Akt)和活性半胱天冬酶9(cleaved caspase-9)蛋白表達(dá)變化。 3.結(jié)果 外源基因轉(zhuǎn)染后表達(dá)特異性蛋白產(chǎn)物。Adv-Mfn2-S442A和Adv-Mfn2誘導(dǎo)細(xì)胞凋亡的作用較對(duì)照組顯著增強(qiáng)(P0.01),線粒體膜電位顯著降低(P0.01),p-Akt表達(dá)水平顯著降低(P0.01),cleaved caspase-9表達(dá)水平顯著增高(P0.01),且Adv-Mfn2-S442A作用更明顯(P0.01),而Adv-Mfn2-S442D組較對(duì)照組無(wú)顯著差異。 4.結(jié)論 去磷酸化的Mfn2通過Akt信號(hào)通路誘導(dǎo)VSMCs凋亡的作用較Mfn2更明顯；而磷酸化的Mfn2無(wú)顯著作用,PKA磷酸化位點(diǎn)的狀態(tài)對(duì)Mfn2調(diào)節(jié)VSMCs凋亡有重要影響。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R341

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