本文選題：骨骼肌　切入點(diǎn)：衛(wèi)星細(xì)胞　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的探討不同年齡小鼠骨骼肌衛(wèi)星細(xì)胞增殖能力的變化,以及熱量限制對(duì)老齡小鼠骨骼肌衛(wèi)星細(xì)胞增殖能力的影響及其機(jī)制。方法本實(shí)驗(yàn)分為三部分,第一部分探討不同年齡小鼠骨骼肌衛(wèi)星細(xì)胞增殖能力的變化。按年齡將C57BL小鼠分為三組:乳鼠(小于1周齡)、成年鼠(3-4月齡)、老年鼠(15-16月齡),分別取后肢骨骼肌以膠原酶及中性酶制備骨骼肌單核細(xì)胞懸液,采用流式細(xì)胞術(shù)(CD45-/CD11b-/Scal-1-/CXCR4+/β1-integrin+分選方案)分選小鼠肌衛(wèi)星細(xì)胞及行細(xì)胞周期測(cè)定;Western blot法檢測(cè)周期蛋白Cyclin A、Cyclin D1、Cyclin E的表達(dá)。第二部分探討熱量限制對(duì)老齡小鼠衛(wèi)星細(xì)胞增殖能力的影響。選取12~13月齡的C57BL雄性小鼠12只,按隨機(jī)數(shù)字表法均分為老年熱量限制組和老年自由飲食組,每組6只,均單籠飼養(yǎng)。實(shí)驗(yàn)開(kāi)始前,2組小鼠均適應(yīng)性飼養(yǎng)2周,計(jì)算平均進(jìn)食量作為老年自由飲食組投食量,約75.09k J/d;老年熱量限制組采用熱量限制法投食,投食量為老年自由飲食組的60%,約45.05k J/d。逐日投放飼料,每周稱(chēng)量體重一次,至實(shí)驗(yàn)第15周結(jié)束。分別比較2組小鼠熱量限制前和熱量限制第15周的體重和骨骼肌指數(shù)變化。分別取后肢骨骼肌制備單核細(xì)胞懸液,采用流式細(xì)胞術(shù)分選小鼠肌衛(wèi)星細(xì)胞,并進(jìn)行細(xì)胞周期測(cè)定;用免疫印跡法檢測(cè)周期蛋白Cyclin A、Cyclin D1、Cyclin E的表達(dá)。第三部分探討熱量限制對(duì)老年小鼠衛(wèi)星細(xì)胞增殖能力影響的可能機(jī)制。AMPK和SIRT1是機(jī)體細(xì)胞和器官中能量代謝相關(guān)蛋白,分別取成年鼠、老年自由飲食組及老年熱量限制組肌肉提取蛋白,通過(guò)蛋白免疫印跡法檢測(cè)AMPKα、p-AMPKα、SIRT1的表達(dá)并比較。結(jié)果與成年鼠[G0/G1期(78.25±0.38)%;S+G2/M期(19.99±1.50)%]相比,乳鼠骨骼肌衛(wèi)星細(xì)胞的G0/G1期(58.89±0.43%)比例降低(P0.01),S期+G2/M期(37.53±0.06%)比例升高(P0.01),Cyclin A(P0.01)、Cyclin E(P0.01)表達(dá)增強(qiáng),Cyclin D1表達(dá)降低(P0.01);老年鼠骨骼肌衛(wèi)星細(xì)胞的G0/G1期(89.78±0.37)比例增高(P0.01),S期+G2/M(10.40±0.70)期比例降低(P0.01);Cyclin A(P0.01)、Cyclin E(P0.01)表達(dá)增強(qiáng),Cyclin D1表達(dá)減弱(P0.05)。熱量限制前,老年熱量限制組和老年自由飲食組小鼠體重分別為(30.7±0.4)g和(30.8±0.3)g,組間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);熱量限制結(jié)束時(shí),老年熱量限制組小鼠的體重降至(19.5±0.4)g,較組內(nèi)熱量限制前明顯降低(P0.001),且明顯低于老年自由飲食組小鼠的體重[(31.9±0.5)g],組間差異有統(tǒng)計(jì)學(xué)意義(P0.001);老年熱量限制組與老年自由飲食組的脛前肌、腓腸肌及伸趾長(zhǎng)肌的骨骼肌指數(shù)差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。與老年自由飲食組[G0/G1期(89.78±0.37)%;S期(4.29±1.57)%]相比,老年熱量限制組骨骼肌衛(wèi)星細(xì)胞的G0/G1期比例[(62.18±0.42)%]下降(P0.001),S期比例(28.75±0.30%)顯著升高(P0.001);Cyclin A(P0.001)、Cyclin E(P0.01)表達(dá)增強(qiáng),Cyclin D1表達(dá)降低(P0.01)。與成年組相比,老年組p-AMPKα/AMPKα和SIRT1表達(dá)顯著降低(P0.01);與老年自由飲食組相比,老年熱量限制組p-AMPKα/AMPKα顯著升高(P0.01)、SIRT1表達(dá)顯著升高(P0.01)。結(jié)論隨年齡的增長(zhǎng),C57BL小鼠骨骼肌衛(wèi)星細(xì)胞增殖能力減弱。熱量限制可以延緩老齡小鼠骨骼肌衛(wèi)星細(xì)胞增殖能力的減退,熱量限制的這種作用與其激活A(yù)MPK/SIRT信號(hào)途徑有關(guān)。
[Abstract]:Objective to investigate the changes of different age of mouse skeletal muscle satellite cell proliferation, and the effect of caloric restriction on aging of mouse skeletal muscle satellite cells proliferation and its mechanism. Methods the experiment was divided into three parts, the first part discusses the change of different age of mouse skeletal muscle satellite cell proliferation. By age C57BL mice were divided into three groups: rat (less than 1 Zhou Ling), adult rats (3-4 months old) and aged rats (15-16 months old), were hindlimb skeletal muscle by collagenase and neutral enzyme preparation of skeletal muscle mononuclear cell suspension by flow cytometry (CD45-/CD11b-/Scal-1-/CXCR4+/ beta 1-integrin+ sorting scheme) separated from mouse muscle satellite cells and the cell cycle determination; detection of cyclin Cyclin A, Western blot Cyclin D1, the expression of Cyclin E. The second part discusses the effect of caloric restriction on aged mice satellite cell proliferation. Select 12~13 months The 12 C57BL mice were randomly divided into elderly group and elderly calorie restriction free diet group, 6 rats in each group were reared in single cage. Before the start of the experiment, 2 groups of mice were fed for 2 weeks, calculate the average intake for the elderly free diet food, about 75.09k J/d; elderly heat limit group with calorie restriction method of feeding, food for the elderly free diet group 60%, about 45.05k J/d. daily feeding, weighed once a week, until the end of the fifteenth week. The 2 groups were compared in mice before calorie restriction and calorie restriction fifteenth week weight and skeletal muscle index changes of hindlimb skeletal muscle respectively. Preparation of mononuclear cell suspension by flow cytometry sorting of mouse muscle satellite cells, and the cell cycle was detected by Western blotting assay; cyclin Cyclin A, Cyclin D1, the expression of Cyclin E. The third part discusses calorie restriction on The possible mechanism of.AMPK and SIRT1 proliferation of satellite cells in aged mice affects the energy metabolism of cells and organs related protein, were obtained from adult rats, free diet group and elderly elderly calorie restriction group of muscle protein extraction, detected by Western blot of AMPK alpha, p-AMPK alpha, SIRT1 expression and compare the results of [G0/G1 and adult. In the period of (78.25 + 0.38)%; S+G2/M (19.99 + 1.50) when compared to rat skeletal muscle satellite cells in G0/G1 phase (58.89 + 0.43%) decreased the proportion of (P0.01) +G2/M, S (37.53 + 0.06%) increased the proportion of Cyclin (P0.01), A (P0.01), Cyclin E (P0.01 the enhanced expression of Cyclin), the decreased expression of D1 (P0.01); aged rat skeletal muscle satellite cells in G0/G1 phase (89.78 + 0.37) proportion increased (P0.01), S +G2/M (10.40 + 0.70) phase was decreased (P0.01); Cyclin A (P0.01), Cyclin E (P0.01) Cyclin expression, D1 expression reduced calorie restriction before (P0.05). ,鑰佸勾鐑噺闄愬埗緇勫拰鑰佸勾鑷敱楗緇勫皬榧犱綋閲嶅垎鍒負(fù)(30.7鹵0.4)g鍜，

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