运动促衰老骨骼肌卫星细胞成肌分化的线粒体重构机制研究

发布时间：2018-05-03 18:19

  本文选题：运动 + 衰老　； 参考：《中国人民解放军军事医学科学院》2012年博士论文

【摘要】：目的：骨骼肌卫星细胞成肌分化能力降低是衰老性肌萎缩发生机制之一,氧化应激是造成衰老卫星细胞成肌分化障碍的重要原因。适量运动训练可降低衰老卫星细胞内氧化应激水平,与成肌分化能力增加相关,但具体机制尚不清楚。研究表明,卫星细胞成肌分化过程也是线粒体重构的过程,且线粒体重构可逆向调控卫星细胞分化能力及定向性。我们前期工作证明,ROS过高产生氧化应激诱导线粒体异常重构,启动病理信号；而运动训练可通过适量上调ROS,发挥其信号分子效应,进而促进线粒体适应性重构,实现健康效应。本研究从ROS生理/病理双重作用角度探讨运动促衰老卫星细胞成肌分化的机制：1)建立衰老性肌萎缩小鼠模型及耐力运动训练干预模型,利用原代培养技术获得骨骼肌卫星细胞,并体外诱导分化,探讨ROS生成、线粒体重构(能量代谢、生物合成、融合分裂、嵴重构)及相关信号通路的变化规律及潜在关联；2)并以C2C12成肌细胞为研究对象,观察成肌分化各时程ROS生成、线粒体重构及相关信号通路的变化规律,以探讨生理条件下它们之间的时序关系；3)对照观察不同水平外源性H202对成肌分化中线粒体重构的影响,进一步验证“运动通过改变ROS水平和线粒体重构调控衰老卫星细胞分化”的假设；并利用mTOR特异性激动剂和阻断剂干预上述模型,探讨mTOR是否参与ROS双向调控成肌分化的切换机制。 方法：1)雄性C57BL/6小鼠48只,包括青年鼠(2月龄,24只)和老年鼠(12月龄,24只)。分为青年对照组、青年运动训练组、老年对照组和老年运动训练组。训练组小鼠进行12周跑台耐力训练。处死后计算腓肠肌湿重/体重比值,并HE染色测量肌纤维横截面积；剩余肌肉用两步酶消化法分离骨骼肌卫星细胞,免疫化学染色鉴定卫星细胞纯度；原代培养并体外诱导成肌分化24h。倒置显微镜观察卫星细胞成肌分化程度；Oxygraph-2k细胞呼吸测量仪测定透膜细胞中线粒体氧耗速率；荧光素-荧光素酶发光法测定ATP合成酶活性；JC-1荧光探针检测线粒体膜电位；DCFH-DA荧光探针检测细胞ROS水平和线粒体ROS生成速率；电镜检测线粒体超微结构；RT-PCR法检测MyHC Ⅰ、MyHC Ⅱa、MyHCⅡx mRNA和MyHCⅡb mRNA表达；Western-blot法检测细胞MyHC、COXⅣ、 Mfn1、OPA1、Drp1、MnSOD、 PGC-lα、AMPK、p-AMPK (Thr172)、mTOR、 p-mTOR (Ser2448)和p2l蛋白表达量；2)用低浓度马血清诱导C2C12成肌细胞分化,分别在成肌分化后0h,3h,6h,9h,12h和24h终止分化,收集细胞,测定成肌分化及线粒体重构相关指标(同前)；3)建立浓度梯度H202干预C2C12成肌细胞分化模型,MTT法检测细胞存活率,RT-PCR法检测MyHC mRNA表达,确定促进成肌分化的低浓度H2O2和抑制成肌分化的高浓度H2O2。在C2C12成肌细胞分化起始时分别加入高或低浓度H2O2,分化24h后检测成肌分化及线粒体重构相关指标(同前)。并在低浓度H2O2干预模型中加入雷帕霉素,在高浓度H2O2干预模型中加入亮氨酸,检测mTOR在ROS调控成肌分化通路中的角色。 结果： 一、运动和衰老对肌卫星细胞分化中线粒体重构和ROS生成的影响 本部分研究中,与青年组比较,老年组肌肉湿重/体重比值和肌纤维横截面积明显降低,表明衰老性肌萎缩模型建立成功。老年组卫星细胞体外分化24h后,肌管形成数量、MyHCⅠ、MyHCⅡa、MyHCⅡx mRNA及MyHC蛋白表达均低于青年组,提示衰老卫星细胞成肌分化能力降低。此外,老年组ST3、RCR、 MnSOD、COXⅣ、 Mfn1、L-OPA1、S-OPA1、Drp1、PGC-lα、Tfam、mTOR、 p-mTOR、p21表达均显著降低；老年组ST4、细胞ROS水平、线粒体ROS生成速率、p-AMPK显著升高。电镜结果显示,老年组线粒体嵴数量极少且排列紊乱。12周运动训练部分逆转了上述衰老对成肌分化、ROS生成、线粒体能量代谢、生物合成、融合分裂及嵴重构的影响。 二、C2C12成肌细胞分化进程中线粒体重构与ROS生成动态变化 本部分研究中,分化3h时,MyHC、ST3、RCR、ATP合成活力、膜电位、细胞ROS、线粒体ROS生成速率、MnSOD、L-OPA1、Mfn1、PGC-1α、 p-mTOR、 p-AMPK和p21即开始显著升高,且升高趋势持续至24h。分化6h时,S-OPA1和(?)nTOR表达开始升高,且升高趋势持续至24h。分化9h时,Tfam表达开始升高,且升高趋势持续至24h。ST4在分化12h和24h时开始升高。COXⅣ在分化24h时才开始明显升高。Drpl在分化6h和9h时显著升高,但在12h时开始降低,至24h时恢复至0h水平。 三、外源性H2O2对C2C12成肌细胞分化中线粒体重构的调控机制研究 在浓度梯度H2O2干预C2C12成肌细胞分化实验中,与Oμmol/L H2O2比较,100μmol/L H2O2对细胞存活率无影响,MyHC mRNA表达升高了31%;600μmol/L H2O2使细胞存活率降低了18%,MyHC mRNA表达降低了28%。我们将100μMH2O2定义促成肌分化的低水平ROS,将600μM H2O2定义抑制成肌分化的高水平ROS。进一步研究表明,与0μmol/L H2O2比较,100μM H2O2使成肌分化、线粒体呼吸速率、ATP合成、膜电位、生物合成、融合分裂、嵴重构均显著上调,加入雷帕霉素抑制甚至逆转了100μM H2O2对成肌分化和线粒体重构的促进作用；600μM H2O2使成肌分化、线粒体呼吸速率、ATP合成、膜电位、生物合成、融合、嵴重构均显著下调,加入亮氨酸部分恢复了600μM H2O2对成肌分化和线粒体重构的抑制作用。 结论： 1.耐力运动训练可通过上调mTOR和PGC-la表达,促进衰老卫星细胞成肌分化中线粒体适应性重构,包括线粒体嵴重构趋向于成熟、线粒体空间结构趋向于融合、线粒体生物合成增加。从而提高线粒体能量代谢水平,继而通过抑制AMPK活化而增加p21表达,从而促进衰老卫星细胞成肌分化；并通过促进MnSOD表达,降低线粒体ROS水平； 2.成肌分化进程中线粒体ROS持续性增加。成肌分化中线粒体重构的可能时序关系：①成肌分化起始时,线粒体嵴重构首先发生以促进线粒体的成熟→②线粒体开始融合形成网络→③线粒体开始分裂并从核周向细胞特定区域移动再定位→④线粒体重新趋于融合→⑤线粒体生物合成开始。提示分化早期细胞能量水平与线粒体自身呼吸速率及空间构象相关,分化早期细胞能量水平与线粒体生物合成相关； 3.低水平H202可通过活化rnTOR和PGC-la,促进衰老卫星细胞成肌分化中线粒体适应性重构,包括线粒体嵴重构趋向成熟、线粒体空间结构趋向融合、线粒体生物合成增加,能量代谢水平增加。本结果与运动作用相似,提示中等负荷耐力运动训练可能通过将ROS保持于一定水平,从而促进线粒体重构和成肌分化；高水平H202可通过抑制mTOR和PGC-1a,导致衰老卫星细胞成肌分化中线粒体重构异常,包括线粒体嵴重构障碍、线粒体空间结构趋向于分裂、线粒体生物合成减少,能量代谢水平降低。本结果与衰老作用相似,提示衰老可能通过高水平ROS抑制线粒体重构和成肌分化；
[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.
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