热量限制延缓老年小鼠骨骼肌卫星细胞增殖能力减退及其机制研究

发布时间：2018-03-14 23:27

  本文选题：骨骼肌　切入点：卫星细胞　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的探讨不同年龄小鼠骨骼肌卫星细胞增殖能力的变化,以及热量限制对老龄小鼠骨骼肌卫星细胞增殖能力的影响及其机制。方法本实验分为三部分,第一部分探讨不同年龄小鼠骨骼肌卫星细胞增殖能力的变化。按年龄将C57BL小鼠分为三组:乳鼠(小于1周龄)、成年鼠(3-4月龄)、老年鼠(15-16月龄),分别取后肢骨骼肌以胶原酶及中性酶制备骨骼肌单核细胞悬液,采用流式细胞术(CD45-/CD11b-/Scal-1-/CXCR4+/β1-integrin+分选方案)分选小鼠肌卫星细胞及行细胞周期测定;Western blot法检测周期蛋白Cyclin A、Cyclin D1、Cyclin E的表达。第二部分探讨热量限制对老龄小鼠卫星细胞增殖能力的影响。选取12~13月龄的C57BL雄性小鼠12只,按随机数字表法均分为老年热量限制组和老年自由饮食组,每组6只,均单笼饲养。实验开始前,2组小鼠均适应性饲养2周,计算平均进食量作为老年自由饮食组投食量,约75.09k J/d;老年热量限制组采用热量限制法投食,投食量为老年自由饮食组的60%,约45.05k J/d。逐日投放饲料,每周称量体重一次,至实验第15周结束。分别比较2组小鼠热量限制前和热量限制第15周的体重和骨骼肌指数变化。分别取后肢骨骼肌制备单核细胞悬液,采用流式细胞术分选小鼠肌卫星细胞,并进行细胞周期测定;用免疫印迹法检测周期蛋白Cyclin A、Cyclin D1、Cyclin E的表达。第三部分探讨热量限制对老年小鼠卫星细胞增殖能力影响的可能机制。AMPK和SIRT1是机体细胞和器官中能量代谢相关蛋白,分别取成年鼠、老年自由饮食组及老年热量限制组肌肉提取蛋白,通过蛋白免疫印迹法检测AMPKα、p-AMPKα、SIRT1的表达并比较。结果与成年鼠[G0/G1期(78.25±0.38)%;S+G2/M期(19.99±1.50)%]相比,乳鼠骨骼肌卫星细胞的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)表达增强,Cyclin D1表达降低(P0.01);老年鼠骨骼肌卫星细胞的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)表达增强,Cyclin D1表达减弱(P0.05)。热量限制前,老年热量限制组和老年自由饮食组小鼠体重分别为(30.7±0.4)g和(30.8±0.3)g,组间差异无统计学意义(P0.05);热量限制结束时,老年热量限制组小鼠的体重降至(19.5±0.4)g,较组内热量限制前明显降低(P0.001),且明显低于老年自由饮食组小鼠的体重[(31.9±0.5)g],组间差异有统计学意义(P0.001);老年热量限制组与老年自由饮食组的胫前肌、腓肠肌及伸趾长肌的骨骼肌指数差异无统计学意义(P0.05)。与老年自由饮食组[G0/G1期(89.78±0.37)%;S期(4.29±1.57)%]相比,老年热量限制组骨骼肌卫星细胞的G0/G1期比例[(62.18±0.42)%]下降(P0.001),S期比例(28.75±0.30%)显著升高(P0.001);Cyclin A(P0.001)、Cyclin E(P0.01)表达增强,Cyclin D1表达降低(P0.01)。与成年组相比,老年组p-AMPKα/AMPKα和SIRT1表达显著降低(P0.01);与老年自由饮食组相比,老年热量限制组p-AMPKα/AMPKα显著升高(P0.01)、SIRT1表达显著升高(P0.01)。结论随年龄的增长,C57BL小鼠骨骼肌卫星细胞增殖能力减弱。热量限制可以延缓老龄小鼠骨骼肌卫星细胞增殖能力的减退,热量限制的这种作用与其激活AMPK/SIRT信号途径有关。
[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�閲忛檺鍒剁粍鍜岃�佸勾鑷�鐢遍ギ椋熺粍灏忛紶浣撻噸鍒嗗埆涓�(30.7卤0.4)g鍜，

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