本文選題：運(yùn)動 + 衰老��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2012年博士論文

【摘要】：目的：骨骼肌衛(wèi)星細(xì)胞成肌分化能力降低是衰老性肌萎縮發(fā)生機(jī)制之一,氧化應(yīng)激是造成衰老衛(wèi)星細(xì)胞成肌分化障礙的重要原因。適量運(yùn)動訓(xùn)練可降低衰老衛(wèi)星細(xì)胞內(nèi)氧化應(yīng)激水平,與成肌分化能力增加相關(guān),但具體機(jī)制尚不清楚。研究表明,衛(wèi)星細(xì)胞成肌分化過程也是線粒體重構(gòu)的過程,且線粒體重構(gòu)可逆向調(diào)控衛(wèi)星細(xì)胞分化能力及定向性。我們前期工作證明,ROS過高產(chǎn)生氧化應(yīng)激誘導(dǎo)線粒體異常重構(gòu),啟動病理信號；而運(yùn)動訓(xùn)練可通過適量上調(diào)ROS,發(fā)揮其信號分子效應(yīng),進(jìn)而促進(jìn)線粒體適應(yīng)性重構(gòu),實(shí)現(xiàn)健康效應(yīng)。本研究從ROS生理/病理雙重作用角度探討運(yùn)動促衰老衛(wèi)星細(xì)胞成肌分化的機(jī)制：1)建立衰老性肌萎縮小鼠模型及耐力運(yùn)動訓(xùn)練干預(yù)模型,利用原代培養(yǎng)技術(shù)獲得骨骼肌衛(wèi)星細(xì)胞,并體外誘導(dǎo)分化,探討ROS生成、線粒體重構(gòu)(能量代謝、生物合成、融合分裂、嵴重構(gòu))及相關(guān)信號通路的變化規(guī)律及潛在關(guān)聯(lián)；2)并以C2C12成肌細(xì)胞為研究對象,觀察成肌分化各時程ROS生成、線粒體重構(gòu)及相關(guān)信號通路的變化規(guī)律,以探討生理?xiàng)l件下它們之間的時序關(guān)系；3)對照觀察不同水平外源性H202對成肌分化中線粒體重構(gòu)的影響,進(jìn)一步驗(yàn)證“運(yùn)動通過改變ROS水平和線粒體重構(gòu)調(diào)控衰老衛(wèi)星細(xì)胞分化”的假設(shè)；并利用mTOR特異性激動劑和阻斷劑干預(yù)上述模型,探討mTOR是否參與ROS雙向調(diào)控成肌分化的切換機(jī)制。 方法：1)雄性C57BL/6小鼠48只,包括青年鼠(2月齡,24只)和老年鼠(12月齡,24只)。分為青年對照組、青年運(yùn)動訓(xùn)練組、老年對照組和老年運(yùn)動訓(xùn)練組。訓(xùn)練組小鼠進(jìn)行12周跑臺耐力訓(xùn)練。處死后計(jì)算腓腸肌濕重/體重比值,并HE染色測量肌纖維橫截面積；剩余肌肉用兩步酶消化法分離骨骼肌衛(wèi)星細(xì)胞,免疫化學(xué)染色鑒定衛(wèi)星細(xì)胞純度；原代培養(yǎng)并體外誘導(dǎo)成肌分化24h。倒置顯微鏡觀察衛(wèi)星細(xì)胞成肌分化程度；Oxygraph-2k細(xì)胞呼吸測量儀測定透膜細(xì)胞中線粒體氧耗速率；熒光素-熒光素酶發(fā)光法測定ATP合成酶活性；JC-1熒光探針檢測線粒體膜電位；DCFH-DA熒光探針檢測細(xì)胞ROS水平和線粒體ROS生成速率；電鏡檢測線粒體超微結(jié)構(gòu)；RT-PCR法檢測MyHC Ⅰ、MyHC Ⅱa、MyHCⅡx mRNA和MyHCⅡb mRNA表達(dá)；Western-blot法檢測細(xì)胞MyHC、COXⅣ、 Mfn1、OPA1、Drp1、MnSOD、 PGC-lα、AMPK、p-AMPK (Thr172)、mTOR、 p-mTOR (Ser2448)和p2l蛋白表達(dá)量；2)用低濃度馬血清誘導(dǎo)C2C12成肌細(xì)胞分化,分別在成肌分化后0h,3h,6h,9h,12h和24h終止分化,收集細(xì)胞,測定成肌分化及線粒體重構(gòu)相關(guān)指標(biāo)(同前)；3)建立濃度梯度H202干預(yù)C2C12成肌細(xì)胞分化模型,MTT法檢測細(xì)胞存活率,RT-PCR法檢測MyHC mRNA表達(dá),確定促進(jìn)成肌分化的低濃度H2O2和抑制成肌分化的高濃度H2O2。在C2C12成肌細(xì)胞分化起始時分別加入高或低濃度H2O2,分化24h后檢測成肌分化及線粒體重構(gòu)相關(guān)指標(biāo)(同前)。并在低濃度H2O2干預(yù)模型中加入雷帕霉素,在高濃度H2O2干預(yù)模型中加入亮氨酸,檢測mTOR在ROS調(diào)控成肌分化通路中的角色。 結(jié)果： 一、運(yùn)動和衰老對肌衛(wèi)星細(xì)胞分化中線粒體重構(gòu)和ROS生成的影響 本部分研究中,與青年組比較,老年組肌肉濕重/體重比值和肌纖維橫截面積明顯降低,表明衰老性肌萎縮模型建立成功。老年組衛(wèi)星細(xì)胞體外分化24h后,肌管形成數(shù)量、MyHCⅠ、MyHCⅡa、MyHCⅡx mRNA及MyHC蛋白表達(dá)均低于青年組,提示衰老衛(wèi)星細(xì)胞成肌分化能力降低。此外,老年組ST3、RCR、 MnSOD、COXⅣ、 Mfn1、L-OPA1、S-OPA1、Drp1、PGC-lα、Tfam、mTOR、 p-mTOR、p21表達(dá)均顯著降低；老年組ST4、細(xì)胞ROS水平、線粒體ROS生成速率、p-AMPK顯著升高。電鏡結(jié)果顯示,老年組線粒體嵴數(shù)量極少且排列紊亂。12周運(yùn)動訓(xùn)練部分逆轉(zhuǎn)了上述衰老對成肌分化、ROS生成、線粒體能量代謝、生物合成、融合分裂及嵴重構(gòu)的影響。 二、C2C12成肌細(xì)胞分化進(jìn)程中線粒體重構(gòu)與ROS生成動態(tài)變化 本部分研究中,分化3h時,MyHC、ST3、RCR、ATP合成活力、膜電位、細(xì)胞ROS、線粒體ROS生成速率、MnSOD、L-OPA1、Mfn1、PGC-1α、 p-mTOR、 p-AMPK和p21即開始顯著升高,且升高趨勢持續(xù)至24h。分化6h時,S-OPA1和(?)nTOR表達(dá)開始升高,且升高趨勢持續(xù)至24h。分化9h時,Tfam表達(dá)開始升高,且升高趨勢持續(xù)至24h。ST4在分化12h和24h時開始升高。COXⅣ在分化24h時才開始明顯升高。Drpl在分化6h和9h時顯著升高,但在12h時開始降低,至24h時恢復(fù)至0h水平。 三、外源性H2O2對C2C12成肌細(xì)胞分化中線粒體重構(gòu)的調(diào)控機(jī)制研究 在濃度梯度H2O2干預(yù)C2C12成肌細(xì)胞分化實(shí)驗(yàn)中,與Oμmol/L H2O2比較,100μmol/L H2O2對細(xì)胞存活率無影響,MyHC mRNA表達(dá)升高了31%;600μmol/L H2O2使細(xì)胞存活率降低了18%,MyHC mRNA表達(dá)降低了28%。我們將100μMH2O2定義促成肌分化的低水平ROS,將600μM H2O2定義抑制成肌分化的高水平ROS。進(jìn)一步研究表明,與0μmol/L H2O2比較,100μM H2O2使成肌分化、線粒體呼吸速率、ATP合成、膜電位、生物合成、融合分裂、嵴重構(gòu)均顯著上調(diào),加入雷帕霉素抑制甚至逆轉(zhuǎn)了100μM H2O2對成肌分化和線粒體重構(gòu)的促進(jìn)作用；600μM H2O2使成肌分化、線粒體呼吸速率、ATP合成、膜電位、生物合成、融合、嵴重構(gòu)均顯著下調(diào),加入亮氨酸部分恢復(fù)了600μM H2O2對成肌分化和線粒體重構(gòu)的抑制作用。 結(jié)論： 1.耐力運(yùn)動訓(xùn)練可通過上調(diào)mTOR和PGC-la表達(dá),促進(jìn)衰老衛(wèi)星細(xì)胞成肌分化中線粒體適應(yīng)性重構(gòu),包括線粒體嵴重構(gòu)趨向于成熟、線粒體空間結(jié)構(gòu)趨向于融合、線粒體生物合成增加。從而提高線粒體能量代謝水平,繼而通過抑制AMPK活化而增加p21表達(dá),從而促進(jìn)衰老衛(wèi)星細(xì)胞成肌分化；并通過促進(jìn)MnSOD表達(dá),降低線粒體ROS水平； 2.成肌分化進(jìn)程中線粒體ROS持續(xù)性增加。成肌分化中線粒體重構(gòu)的可能時序關(guān)系：①成肌分化起始時,線粒體嵴重構(gòu)首先發(fā)生以促進(jìn)線粒體的成熟→②線粒體開始融合形成網(wǎng)絡(luò)→③線粒體開始分裂并從核周向細(xì)胞特定區(qū)域移動再定位→④線粒體重新趨于融合→⑤線粒體生物合成開始。提示分化早期細(xì)胞能量水平與線粒體自身呼吸速率及空間構(gòu)象相關(guān),分化早期細(xì)胞能量水平與線粒體生物合成相關(guān)； 3.低水平H202可通過活化rnTOR和PGC-la,促進(jìn)衰老衛(wèi)星細(xì)胞成肌分化中線粒體適應(yīng)性重構(gòu),包括線粒體嵴重構(gòu)趨向成熟、線粒體空間結(jié)構(gòu)趨向融合、線粒體生物合成增加,能量代謝水平增加。本結(jié)果與運(yùn)動作用相似,提示中等負(fù)荷耐力運(yùn)動訓(xùn)練可能通過將ROS保持于一定水平,從而促進(jìn)線粒體重構(gòu)和成肌分化；高水平H202可通過抑制mTOR和PGC-1a,導(dǎo)致衰老衛(wèi)星細(xì)胞成肌分化中線粒體重構(gòu)異常,包括線粒體嵴重構(gòu)障礙、線粒體空間結(jié)構(gòu)趨向于分裂、線粒體生物合成減少,能量代謝水平降低。本結(jié)果與衰老作用相似,提示衰老可能通過高水平ROS抑制線粒體重構(gòu)和成肌分化；
[Abstract]:Objective: the decrease of the differentiation ability of skeletal muscle satellite cells is one of the mechanisms of aging muscular atrophy. Oxidative stress is an important cause of the dysfunction of senescent satellite cells. Appropriate exercise training can reduce the level of oxidative stress in senescent satellite cells, which is related to the increase of the ability of myogenic differentiation, but the specific mechanism is not yet clear. The study shows that the process of the differentiation of the satellite cells is also the process of mitochondrial reconfiguration, and the mitochondrial reconfiguration can reverse the differentiation ability and directionality of the satellite cells. Our previous work has proved that ROS excessively high oxidative stress induced abnormal remodeling of mitochondria and starting the pathological signal; and exercise training can be used to signal its signal through a proper amount of ROS to play its signal. Molecular effects, which further promote the adaptive remodeling of mitochondria and achieve health effects. This study explores the mechanism of myogenic differentiation of aging satellite cells from the dual role of ROS and pathology: 1) to establish an aging model of amyotrophic mice and an intervention model of endurance exercise training, and to obtain skeletal muscle satellite cells by using the primary culture technique. In vitro differentiation, the changes of ROS formation, mitochondrial reconstruction (energy metabolism, biosynthesis, fusion division, crista reconstruction) and related signal pathways were investigated. 2) C2C12 myoblasts were used as the research object to observe the generation of ROS, reconfiguration of linear grain body and the changes of related signal pathways. The time series relationship between them; 3) to observe the effect of different levels of exogenous H202 on mitochondrial remodeling in the differentiation of myogenic differentiation, and further verify the hypothesis that "exercise regulates senescence satellite cell differentiation by changing ROS level and mitochondrial reconfiguration", and using mTOR specific agonists and blockers to intervene the above model and explore mT Whether OR participates in the mechanism of ROS bidirectional regulation of myogenic differentiation.
Methods: 1) 48 male C57BL/6 mice, including young rats (2 month old, 24) and old rats (12 month old, 24), were divided into young control group, young exercise training group, old control group and old exercise training group. The training group was trained for 12 weeks' endurance training. After death, the gastrocnemius wet weight / weight ratio was calculated, and HE staining was used to measure the muscle fiber transverse. The skeletal muscle satellite cells were separated by two step enzyme digestion method, and the purity of the satellite cells was identified by immunochemistry staining. The degree of myogenic differentiation of the satellite cells was observed by the primary culture and in vitro induction of the myogenic differentiation 24h. inverted microscope. The oxygen consumption rate of the mitochondria in the permeable cells was measured by the Oxygraph-2k cell respiration measuring instrument. The activity of ATP synthase was measured by the luminescence luciferase method, the JC-1 fluorescence probe was used to detect the mitochondrial membrane potential, and the DCFH-DA fluorescence probe was used to detect the ROS level and the formation rate of mitochondrial ROS, and the ultrastructure of the mitochondria was detected by the electron microscope; the RT-PCR method was used to detect MyHC I, MyHC II A, MyHC II x mRNA and ROS Cell MyHC, COX IV, Mfn1, OPA1, Drp1, MnSOD, PGC-l alpha, AMPK, p-AMPK (Thr172), mTOR, p-mTOR (Thr172), p-mTOR, and protein expression; 2) differentiate into myoblast with low concentration horse sera and differentiate after differentiation, collect cells, determine myogenic differentiation and mitochondrial remodeling related indicators (Tong Qian); 3) A concentration gradient H202 was established to interfere with C2C12 myoblast differentiation model, MTT method was used to detect cell viability, RT-PCR method was used to detect the expression of MyHC mRNA. The low concentration of H2O2 and high concentration H2O2. to inhibit the differentiation of myoblasts were determined by adding high or low concentration H2O2 respectively at the beginning of the differentiation of myoblast, and the differentiation and line of the myoblasts were detected after the differentiation. In the low concentration H2O2 intervention model, rapamycin was added to the low concentration H2O2 intervention model, and leucine was added to the high concentration H2O2 intervention model to detect the role of mTOR in the ROS regulation of the myogenic differentiation pathway.
Result錛，

本文編號：1839568