【摘要】：骨骼肌為主要的運(yùn)動應(yīng)答器官,運(yùn)動中骨骼肌對能量的需求不斷地變化,線粒體在應(yīng)答運(yùn)動應(yīng)激引起的氧耗、ATP需求增加的同時,自身形態(tài)、結(jié)構(gòu)、功能都發(fā)生著積極適應(yīng)變化,運(yùn)動促進(jìn)線粒體生物發(fā)生、提高線粒體功能的同時還促進(jìn)細(xì)胞自噬、線粒體自噬等逆向適應(yīng),加速細(xì)胞新陳代謝和線粒體網(wǎng)絡(luò)的更新。線粒體是高度動態(tài)化細(xì)胞器,不斷地進(jìn)行著融合、分裂,線粒體融合、分裂不但在調(diào)控線粒體形態(tài)、功能以及對凋亡信號的應(yīng)答中扮演著重要的角色,還與線粒體生物發(fā)生和線粒體自噬過程緊密偶聯(lián)。MFN2不但調(diào)控線粒體外膜的融合,還調(diào)控線粒體與內(nèi)質(zhì)網(wǎng)的相互作用,影響線粒體對Ca~(2+)的攝取和細(xì)胞能量代謝。MFN2還在E3泛素連接酶Parkin介導(dǎo)的線粒體自噬中起重要作用,MFN2缺失抑制了Parkin向功能紊亂線粒體的定位；同時,MFN2缺失還提高了PGC-1α的表達(dá),提高線粒體生物發(fā)生。MFN2在運(yùn)動調(diào)控骨骼肌線粒體生理適應(yīng)中的作用尚不清楚,本研究著重從線粒體生物發(fā)生、線粒體自噬和線粒體融合、分裂三個角度探索阻斷MFN2對運(yùn)動訓(xùn)練調(diào)控骨骼肌線粒體運(yùn)動適應(yīng)的影響和相關(guān)分子機(jī)制。目的：本文選取MCK-Cre MFN2~(flox/flox)小鼠為研究對象,探索MFN2在骨骼肌線粒體自噬和線粒體生物發(fā)生中的作用,探索阻斷MFN2對運(yùn)動調(diào)控骨骼肌線粒體生理適應(yīng)的影響,主要通過線粒體生物發(fā)生、線粒體自噬和線粒體融合、分裂三個角度探討運(yùn)動調(diào)控骨骼肌線粒體生理適應(yīng)的機(jī)制。方法：通過Cre/Loxp技術(shù)構(gòu)建條件性骨骼肌MFN2基因敲除小鼠及同窩對照小鼠,基因型分別為MCK-Cre MFN2~(flox/flox)和MFN2~(flox/flox)。1)動物水平對MFN2基因與骨骼肌線粒體自噬和線粒體生物發(fā)生的關(guān)系進(jìn)行探索。實(shí)驗(yàn)分組：3月齡雄性MCK-Cre MFN2~(flox/flox)小鼠和同窩MFN2~(flox/flox)小鼠分別作為基因敲除組(KKnockout group, K, n=24)和正常對照組(Control group, C, n=24)。每組隨機(jī)選取8只小鼠分離雙側(cè)股四頭肌、腓腸肌和脛骨前肌,左側(cè)用于RT-qPCR實(shí)驗(yàn),右側(cè)用于Western blotting實(shí)驗(yàn)；每組再隨機(jī)選取8只小鼠,分離雙側(cè)腓腸肌和股四頭肌,左側(cè)4%多聚甲醛固定,制作石蠟切片用于HE染色和免疫組織化學(xué)指標(biāo)檢測,右側(cè)2.5%戊二醛電鏡固定液固定,用于電鏡檢測線粒體形態(tài)、自噬小體等；每組再隨機(jī)選取8只小鼠,分離雙側(cè)腓腸肌用于提取線粒體,檢測線粒體膜電位、檸檬酸酶(CS)活性、H_2O_2釋放,部分樣品用于骨骼肌氧化應(yīng)激指標(biāo)檢測。2)動物水平對MFN2與骨骼肌線粒體運(yùn)動適應(yīng)的關(guān)系進(jìn)行探索。實(shí)驗(yàn)分組：2月齡MCK-Cre MFN2~(flox/flox)小鼠和同窩MFN2~(flox/flox)小鼠隨機(jī)分為正常對照組(Control group,C, n=8)、正常運(yùn)動組(Exercise group, E, n=8)、基因敲除組(Knockout group,K, n=8)、基因敲除運(yùn)動組(Knockout Exercise group, KE, n=8)。 E和KE組小鼠進(jìn)行4周耐力跑臺訓(xùn)練,跑速為14m/min, C和K組置于靜止跑臺,自由活動,最后一次運(yùn)動訓(xùn)練結(jié)束12h處死小鼠分離雙側(cè)腓腸肌,左側(cè)用于RT-qPCR實(shí)驗(yàn),右側(cè)用于Western blotting口氧化應(yīng)激指標(biāo)檢測。本研究檢測指標(biāo)具體如下：1)代謝相關(guān)指標(biāo)：體重、附睪脂肪含量及體質(zhì)百分比、肝臟和心臟濕重。2)線粒體融合、分裂相關(guān)指標(biāo)：RT-qPCR檢測MFN2、MFN1、Drp1、FIS1、OPA1、 MFF mRNA表達(dá)；Western blotting檢測MFN2、MFN1、OPA1、Drp1、MFF蛋白表達(dá)。3)細(xì)胞自噬和線粒體自噬相關(guān)指標(biāo)：RT-qPCR檢測Atg5、Atg12、ULK1、Parkin、PINK1. LC3、p62、BNIP3、FUNDC1、LAMP2 mRNA表達(dá)；Western blotting檢測ULK1、Parkin、 PINK1、LC3、BNIP3、p62、Atg5、AMPKa蛋白表達(dá)以及AMPKa Thr~(172)和ULK1Ser~(555)磷酸化水平。4)線粒體生物發(fā)生相關(guān)指標(biāo)：RT-qPCR檢測PGC-1α、TFAM、NRF1、TFBIM、ATP5a. COXI、COX5b. NDUFS8 mRNA表達(dá),mtDNA含量；Western blotting檢測PGC-1α、 TFAM、NRF1、TOM20蛋白表達(dá)。5)線粒體功能指標(biāo)：檢測線粒體膜電位、CS活性,免疫組化COX和SDH染色。6)形態(tài)學(xué)指標(biāo)：HE染色觀察肌纖維形態(tài)和橫截面積,電鏡檢測肌纖維形態(tài)、線粒體形態(tài)以及自噬小體等。7)氧化應(yīng)激指標(biāo)：檢測離體線粒體H_2O_2釋放和在體骨骼肌H_2O_2和MDA含量,T-SOD活性。結(jié)果：1)通過Cre/Loxp技術(shù)建立了骨骼肌特異性MFN2基因敲除小鼠模型,骨骼肌MFN2 mRNA和蛋白含量較比對照組均顯著下降(p0.01)。2)與C組相比,K組小鼠骨骼肌中異常線粒體增加,部分線粒體出現(xiàn)腫脹,有的嵴折疊下降,甚至出現(xiàn)不規(guī)則的空泡；小鼠骨骼肌Drp1蛋白含量顯著提高(p0.05)。與C組相比,K組小鼠骨骼肌線粒體膜電位顯著下降(p0.05),線粒體H_2O_2釋放率顯著高于C組(p0.05),骨骼肌H_2O_2和MDA含量均顯著高于C組(p0.05)。與C組相比,K組小鼠骨骼肌中PGC-1α、TFAM和線粒體標(biāo)志物TOM20蛋白含量顯著提高(p0.05)。3)與C組相比,K組小鼠骨骼肌FUNDC1、BNIP3 mRNA表達(dá)顯著提高(p0.05),BNIP3、 Parkin、p62蛋白含量顯著增加(p0.05), LC3-Ⅱ/Ⅰ比值顯著提高(p0.05), AMPKαThr~(172)和ULK1Ser~(555)位點(diǎn)磷酸化水平顯著下降(p0.05),肌纖維中自噬小體堆積。4)與C組相比,E組小鼠骨骼肌MFN1、MFN2、Drp1、FIS1mRNA表達(dá)顯著提高(p0.05),MFN1、Drp1蛋白含量顯著提高(p0.05)；與K組相比,KE組小鼠MFN1、MFN2、Drp1 FIS1mRNA表達(dá)顯著提高(p0.05), MFN1、Drp1蛋白含量顯著提高(p0.05)；KE組小鼠骨骼肌Drp1蛋白含量顯著高于E組(p0.05)。5)與C組相比,E組小鼠骨骼肌PGC-1α、NRF1、TFAM、TFB1M、COXI、COX5b mRNA表達(dá)顯著提高(P0.05或P0.01),ntDNA含量、PGC-1α、TFAM 和 TOM20蛋白含量顯著提高(P0.05)；與K組相比,KE組小鼠骨骼肌PGC-1α、NRF1、TFAM、TFB1M、 COXI、COX5bmRNA表達(dá)顯著提高(P0.05或P0.01), PGC-1α、TFAM和TOM20蛋白含量顯著提高(P0.05)；KE組小鼠骨骼肌TFAM蛋白含量顯著高于E組(P0.05)。6)與C組相比,E組小鼠骨骼肌LC3、Atg5、ULK1、BNIP3、Parkin、FUNDC1 mRNA表達(dá)顯著提高(P0.05或P0.01),LC3-II/I匕值顯著提高(P0.05), Parkin、PINK1、BNIP3.ULKl蛋白含量顯著提高(P0.05), AMPKαThr~(172)、ULK1 Ser~(555)位點(diǎn)的磷酸化顯著提高(P0.05)；與E組相比,KE組小鼠骨骼肌LC3、Atg5、ULK1、BNIP3、FUNDC1 mRNA表達(dá)顯著提高(P0.05),LC3-Ⅱ/Ⅰ比值、p62、PINK1、ULK1蛋白含量沒有顯著變化,Parkin、BNIP3、AMPKαThr~(172)、ULK1 Ser~(555)位點(diǎn)的磷酸化顯著提高(P0.05)；KE組小鼠骨骼肌BNIP3、FUNDC1 mRNA表達(dá)以及Parkin、BNIP3、p62蛋白含量顯著高于E組(p0.05)。7)與C組相比,E組小鼠T-SOD活性顯著提高(p0.05),MDA顯著降低(p0.05)；與K組相比,KE組小鼠T-SOD活性顯著提高(p0.05)；KE組小鼠骨骼肌H2O2、MDA顯著高于E組(p0.05或p0.01)。結(jié)論：1)MFN2缺失擾亂了骨骼肌線粒體融合、分裂動態(tài)平衡,引起異常形態(tài)線粒體增加,提高線粒體活性氧的產(chǎn)生和骨骼肌氧化應(yīng)激。2)MFN2缺失提高了骨骼肌線粒體生物發(fā)生,有利于維持mtDNA含量和線粒體呼吸鏈功能。3)MFN2缺失抑制了骨骼肌細(xì)胞自噬和Parkin介導(dǎo)線粒體自噬,但代償性提高了BNIP3依賴的線粒體自噬途徑。4)耐力訓(xùn)練建立更高水平的骨骼肌線粒體融合、分裂,伴隨著線粒體生物發(fā)生、細(xì)胞自噬和線粒體自噬的提高,提示運(yùn)動訓(xùn)練對調(diào)控骨骼肌線粒體質(zhì)量控制有積極作用,MFN2的缺失雖然抑制了運(yùn)動訓(xùn)練誘導(dǎo)的骨骼肌細(xì)胞自噬和Parkin介導(dǎo)的線粒體自噬,但進(jìn)一步提高了線粒體生物發(fā)生和BNIP3介導(dǎo)的線粒體自噬。5)耐力訓(xùn)練并不能通過激活A(yù)MPK-ULK1信號通路改善MFN2缺失小鼠骨骼肌細(xì)胞自噬水平。
[Abstract]:Skeletal muscle is the main motor response organ. The energy requirement of skeletal muscle is constantly changing during exercise. Mitochondria respond to the oxygen consumption and ATP requirement caused by exercise stress. At the same time, their morphology, structure and function have been actively adapted to changes. Exercise promotes mitochondrial biogenesis, improves mitochondrial function and promotes cells at the same time. Autophagy, mitochondrial autophagy and other adverse adaptation, accelerate cell metabolism and mitochondrial network renewal. Mitochondria are highly dynamic organelles, constantly fusing, dividing, mitochondrial fusion, division not only in the regulation of mitochondrial morphology, function and response to apoptotic signals play an important role, but also with mitochondrial organisms. MFN2 not only regulates the membrane fusion of mitochondria, but also the interaction between mitochondria and endoplasmic reticulum. MFN2 also affects the uptake of Ca ~ (2+) by mitochondria and cellular energy metabolism. MFN2 also plays an important role in E3 ubiquitin ligase Parkin-mediated mitochondrial autophagy. MFN2 deletion inhibits Parkin-directed functional turbulence. At the same time, the deletion of MFN2 also increased the expression of PGC-1a and mitochondrial biogenesis. The role of MFN2 in regulating the physiological adaptation of skeletal muscle mitochondria is still unclear. This study focused on mitochondrial biogenesis, mitochondrial autophagy and mitochondrial fusion, and mitosis to explore the effect of blocking MFN2 on exercise training regulation. Objective: To investigate the effects of MFN2 on mitochondrial autophagy and mitochondrial biogenesis in skeletal muscle of MCK-Cre MFN2 ~ (flox/flox) mice, and to explore the effects of blocking MFN2 on mitochondrial adaptation to exercise, mainly through mitochondria. Methods: The conditioned skeletal muscle MFN2 knockout mice and the homologous control mice were constructed by Cre/Loxp technique. The genotypes were MCK-Cre MFN2~ (flox/flox) and MFN2~ (flox/flox). 1) at animal level. The relationship between MFN2 gene and skeletal muscle mitochondrial autophagy and mitochondrial biogenesis was explored. The experimental groups were divided into 3-month-old male MCK-Cre MFN2~ (flox/flox) mice and the same litter MFN2~ (flox/flox) mice as gene knockout group (KKnockout group, K, n=24) and normal control group (control group, C, n=24). The left quadriceps femoris, gastrocnemius and anterior tibial muscles were used for RT-q PCR and the right for Western blotting. Eight mice in each group were randomly selected to separate the bilateral gastrocnemius and quadriceps femoris. The left quadriceps femoris were fixed with 4% paraformaldehyde, and paraffin sections were made for HE staining and immunohistochemical detection. Fixed solution was used for electron microscopic examination of mitochondrial morphology and autophagy, and 8 mice in each group were randomly selected to isolate bilateral gastrocnemius muscle for extraction of mitochondria, detection of mitochondrial membrane potential, citrate enzyme (CS) activity, H_2O_2 release, and some samples for detection of skeletal muscle oxidative stress indicators. 2) Animal level of MFN2 and skeletal muscle mitochondria mitochondria. The experimental group: 2-month-old MCK-Cre MFN2~ (flox/flox) mice and the same litter MFN2~ (flox/flox) mice were randomly divided into normal control group (control group, C, n = 8), normal exercise group (Exercise group, E, n = 8), gene knockout group (Knockout group, K, n = 8), gene knockout exercise group (Knockout Exercise group, KE, n = 8). 8) The mice in E and KE groups were trained on the treadmill for 4 weeks at a running speed of 14 m/min. The mice in C and K groups were placed on the stationary treadmill to move freely. At the end of the last exercise training, the mice were sacrificed to separate bilateral gastrocnemius muscles. The left side was used for RT-qPCR test, and the right side was used for the detection of oxidative stress in Western blotting mouth. Relevant indicators: body weight, epididymal fat content and body mass percentage, liver and heart wet weight. 2) Mitochondrial fusion, mitosis related indicators: RT-qPCR detection of MFN2, MFN1, Drp1, FIS1, OPA1, MFF mRNA expression; Western blotting detection of MFN2, MFN1, OPA1, Drp1, MFF protein expression. 3) cell autophagy and mitochondrial autophagy related indicators: RT-qPCR detection of Atg5, Drp1, MFF protein expression. Atg12, ULK12, ULK1, Parkin, PINK1.LC3, p62, BNIP3, BNIP3, FUNDC1, FUNDC1, LAMP2 mRNAexpression; Western blotdetection ULK1, Parkin, PINK1, LC3, BNIP3, p62, Atg5, AMPKa protein expression and AMPKa Thr ~ (172) and ULK1 Ser ~ (555) phosphophosphorylation levels. 4) Mitochondriabiogenerelated indicators: RT-qPCR detection of PGC-1alpha, TFAM, TFAM, NRF1, NRF1, TFBIM, TFBIM, LCP5a, COCOCOP5a. COCOCOA. COCOCOA. COXFS8.5, COX 8.RN Mitochondrial function index: Mitochondrial membrane potential, CS activity, immunohistochemical COX and SDH staining. 6) Morphological index: HE staining to observe muscle fiber morphology and cross-sectional area, electron microscopy to detect muscle fiber morphology, mitochondrial morphology and autophagy. 7) Oxidative stress index: The release of H_2O_2 from mitochondria in vitro, the content of H_2O_2 and MDA in skeletal muscle in vivo, and the activity of T-SOD were detected. Results: 1) The skeletal muscle-specific MFN2 knockout mice model was established by Cre/Loxp technique. The content of MFN2 mRNA and protein in skeletal muscle of K group was significantly lower than that of control group (p0.01). The content of Drp1 protein in skeletal muscle of mice was significantly increased (p0.05). Compared with group C, the mitochondrial membrane potential of skeletal muscle of mice in group K was significantly decreased (p0.05), the release rate of H_2O_2 in mitochondria was significantly higher than that of group C (p0.05). Compared with group C, the contents of PGC-1a, TFAM and mitochondrial marker TOM20 protein in skeletal muscle of mice in group K were significantly increased (p0.05). Compared with group C, the expressions of FUNDC1 and BNIP3 mRNA in skeletal muscle of mice in group K were significantly increased (p0.05), the contents of BNIP3, Parkin and p62 protein were significantly increased (p0.05), and the ratio of LC3-II/I was significantly increased (p0.05). Compared with group C, the expression of MFN1, MFN2, Drp1, FIS1 mRNA in skeletal muscle of mice in group E was significantly increased (p0.05), and the expression of MFN1, MFN2, and P1 FIS1 mRNA in skeletal muscle of mice in group K was significantly increased (p0.05). Compared with group C, the expression of PGC-1a, NRF1, TFAM, TFB1M, COXI, COX5b mRNA in skeletal muscle of mice in group E increased significantly (P 0.05 or P 0.01), and the content of ntDNA, PGC-1a, TFAM and TOM20 protein in group K increased significantly (P 0.05). Compared with KE group, the expressions of PGC-1a, NRF1, TFAM, TFB1M, COXI, COX5b mRNA in skeletal muscle of mice in KE group were significantly increased (P 0.05 or P 0.01), and the contents of PGC-1a, TFAM and TOM20 protein were significantly increased (P 0.05); the contents of TFAM protein in skeletal muscle of mice in KE group were significantly higher than those in E group (P 0.05). LC3-II/I dagger value was significantly increased (P 0.05), Parkin, PINK1, BNIP 3.ULKl protein content was significantly increased (P 0.05), AMPK alpha Thr ~ (172), ULK1 Ser ~ (555) phosphorylation was significantly increased (P 0.05), compared with E group, the expression of LC3, Atg5, ULK1, BNIP 3, FUNDC 1 mRNA in skeletal muscle of KE group was significantly increased (P 0.05), LC3-II/I ratio, p62, p62, ULK1 Ser ~ (555). PINK1, ULK1 protein content did not change significantly, Parkin, BNIP3, AMPK alpha Thr ~ (172), ULK1 Ser ~ (555) site phosphorylation significantly increased (P 0.05); KE group mice skeletal muscle BNIP3, FUNDC1 mRNA expression and Parkin, BNIP3, p62 protein content significantly higher than E group (p 0.05). 7 compared with C group, E group mice T-SOD activity significantly increased (p 0.05), MDA significantly decreased (p 0.05). Compared with K group, the activity of T-SOD in KE group was significantly increased (p0.05); H2O2 and MDA in skeletal muscle of KE group were significantly higher than those of E group (p0.05 or p0.01). Conclusion: 1) MFN2 deficiency disturbed mitochondrial fusion, mitotic homeostasis, increased abnormal mitochondrial morphology, increased production of reactive oxygen species and oxidative stress in skeletal muscle. Loss of MFN2 inhibits autophagy of skeletal muscle cells and mitochondrial autophagy mediated by Parkin, but compensates for BNIP3-dependent mitochondrial autophagy pathway. 4) Endurance training establishes a higher level of mitochondrial fusion and division in skeletal muscle. With the development of mitochondrial biogenesis, autophagy and mitochondrial autophagy increased, suggesting that exercise training plays an active role in regulating the quality control of skeletal muscle mitochondria. Although the deletion of MFN2 inhibits the autophagy of skeletal muscle cells induced by exercise training and the autophagy of mitochondria mediated by Parkin, it further improves mitochondrial biogenesis and BNIP3. Mediated mitochondrial autophagy.5) Endurance training can not improve the autophagy level of skeletal muscle cells in MFN2-deficient mice by activating AMPK-ULK1 signaling pathway.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：G804.2
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本文編號：2226335