【作者】 刘子青；

【导师】 袁海平；

【作者基本信息】 上海体育学院 ， 运动人体科学， 2010， 硕士

【摘要】 研究目的：骨骼肌卫星细胞是骨骼肌中具有分化增殖潜能的肌源性干细胞,在骨骼肌的生长、创伤修复和运动性骨骼肌的适应性肥大方面发挥重要的作用。IGF-1已被证实在骨骼肌的肥大过程中发挥重要的调节作用,可以促进离体培养的骨骼肌卫星细胞的增殖和分化。MGF (Mechano growth factor)是近年来发现的IGF-1的异构体,是惟一在运动训练和肌肉损伤后显著上调的因子,且具有促进肌卫星细胞增殖的功能。外源性MGF干预是否能够促进骨骼肌卫星细胞增殖,目前尚不明确。鉴于此目的,本研究且通过离体培养骨骼肌卫星细胞,通过不同剂量的MGF干预,探讨对卫星细胞增殖的影响及相关的信号通路中蛋白的表达,为进一步摸索MGF促进肌肉生长的机制,同时也为MGF在临床和运动实践中的应用提供实验依据。研究方法4周龄雄性SD鼠一只,约100g,无菌条件下分离大鼠腿部、背部肌肉,Ⅱ型胶原酶和胰酶两步酶消化法分离骨骼肌卫星细胞,差速贴壁法纯化细胞,用结蛋白(desmin)免疫组织化学进行细胞的鉴定。调整细胞密度,将细胞按MGF浓度分为Ong/ml、25ng/ml、50ng/ml、100ng/ml和150ng/ml五组,用CCK-8检测肌卫星细胞的增殖情况；BCA法测定各组卫星细胞中总蛋白的表达情况；Western Blot检测骨骼肌卫星细胞Akt及p-Akt蛋白的表达情况。研究结果1、肌卫星细胞原代培养：刚分离出来的卫星细胞圆形,折光性强。48小时后贴壁完全,细胞形状不规则,可见大小突起；4天后,细胞之间可见突起连接,细胞呈扁梭形,细胞密度增加。6天后细胞之间相互产生连接,部分细胞分化为肌管。2、肌卫星细胞增殖情况：与对照组相比,25ng/ml组和50ng/ml组在一定时间范围内促进骨骼肌卫星细胞的增殖(P<0.05)。25ng/ml组在24小时到96小时内显著促进细胞增殖(P<0.01)。50ng/ml在24小时到72小时内显著促进细胞增殖(P<0.01)。而100ng/ml和150ng/ml组与对照组相比没有显著差异。从不同时间段来看,与0小时相比,第24小时阶段仅25ng/ml组有高度显著性差异(P<0.01)；第48小时和72小时阶段,25ng/ml和50ng/ml组具有高度显著性差异差异(P<0.01)；特别是在72小时,两组的OD值均达到最高。在96小时时,25ng/ml组与0小时时相比显著性具有差异(P<0.05),其他组无差异。肌卫星细胞总蛋白表达情况：骨骼肌卫星细胞BCA测定结果显示,25ng/ml组总蛋白含量显著高于对照组(P<0.01)；50ng/ml组与对照组相比差异具有显著性(P<0.05)。3、Western Bolt检测结果显示,p-Akt、Akt和GADPH在60 kD和36 kD表达,免疫印迹条带清晰。25ng组和50ng组p-Akt/Akt表达明显高于对照组,差异具有显著性(P<0.01,P<0.05)。100ng组和150ng组与对照组相比没有变化。结论：1. Desmin抗体鉴定表明本研究所采用的Ⅱ型胶原酶和胰蛋白酶两步消化加两次重复差速贴壁的方法成功地获取了骨骼肌卫星细胞。2.MGF干预卫星细胞后发现,25ng/ml和50ng/ml的MGF对骨骼肌卫星细胞具有很好的促增殖作用。3. Western Bolt结果显示,25ng/ml和50ng/ml组Akt和p-Akt的表达上调,MGF诱导的肌卫星细胞的增殖可能是通过Akt信号转导通路。更多还原

【Abstract】 PurposeSkeletal muscle satellite cells are kind of muscle-derived stem cells with potential ability of differentiation and proliferation. Activated satellite cells can keep continued proliferation, differentiation, and ultimately fusing in the damage muscle or generating new muscle fibers. IGF-1 has been confirmed playing an important role in the regulation of skeletal muscle hypertrophy. More and more researches found that MGF, one of the isomers of IGF-1, increased significantly after exercise and muscle injury. And it can promote the cultured skeletal muscle satellite cell proliferation and differentiation in vitro. However, we still don’t know the proper concentration of MGF on skeletal muscle satellite cell proliferation in vitro. And on the research of the mechanism of proliferation of satellite, more focus now is paid on the Akt signal transduction pathway. In the study, we cultured the rat skeletal muscle satellite cells in vitro and supplied them with five different concentrationes of MGF to find the proper concentration of MGF. Then we used the method of Western Blot to investigate the expression of Akt in all five groups.MethodsWe isolated the leg and the back muscles of a 4-week-old male SD rat in sterile conditions. Satellite cells were obtained by two-step digestion method and velocity sedimentation method, seed in cell culture bottle. Then the anti-desmin was used to identify the satellite cells. Adjust the density; we were divided the satellite cells into five groups according to the concentration of MGF:Ong/ml,25ng/ml, 50ng/ml, 100ng/ml and 150ng/ml. The cells were incubated 96h with different concentration of MGF. A method of CCK-8 was used to test the proliferation of the five groups every 24 hours. The total protein content of the satellite cells was test by the method of BCA. Akt and p-Akt was tested by the method of Western Blotting.Results1. Muscle satellite cells primary culture:after isolated, satellite cells scattered in the bottom in the shape of round and have a strong cell refractive index. 24 hours later, the majority of cells were adhered and the adhesion completed in 48 hours. By then, the adherent cells were irregular in shape, and protruding could be seen in the microscope. The cells connection can be seen in the 96 hours. And the cells are in the shape of spindle. The cell density increased, too. In 144 (6d) hours, some cells differentiated into myotubes. Anti-desmin showed that the purity of muscle satellite cells was 95%. And the cells could be used for following research.2. Satellite cell proliferation test:compared with the control group,25ng/ml and 50ng/ml groups promoted skeletal muscle satellite cell proliferation within a certain time, especially the 25ng/ml group could promote cell proliferation within 24h to 96h (P<0.01) significantly. While the 50ng/ml group enhanced cell proliferation within 24h to 72h (P<0.01). However, both the 100ng/ml and 150ng/ml groups had no change compared with the control group.Viewed from the different time points:compared with Oh, only the 25ng/ml group showed significant increase in the first 24h stage (P<0.01). While between the 48h and 72h stage, both 25ng/ml and 50ng/ml groups had a great increase (P <0.01), particularly in the 72h stage, the two groups reached the highest OD value. In the 96h stage, only the 25ng/ml group increased (P<0.05), but had a less OD than the 72h stage.The BCA test showed that 25ng/ml group had a significantly higher total protein content than the control group (P<0.01). While the 50ng/ml group also increased compared with the control group (P<0.05). And the results were consistent with Western Blot.The Akt and p-Akt results had a significant raise in 25ng/ml (P<0.01) and 50ng/ml (P<0.05) group compared with the control group.Conclusions1. Two-step digestion and velocity sedimentation method was used to isolate skeletal muscle satellite cells. Anti-desmin test showed that the methods were successful for the skeletal muscle satellite cells release.2. In the study, concentrations of 25ng/ml and 50ng/ml of MGF on skeletal muscle satellite cells had a great promotional effect on the proliferation. And MGF-induced muscle satellite cell proliferation might be through the Akt signaling pathway.更多还原