MGF对骨骼肌卫星细胞激活的影响及与其增殖的量效关系
发布时间：2018-11-23 11:31
【摘要】：研究目的 在骨骼肌生长、发育、损伤和修复过程中,骨骼肌卫星细胞起着重要的作用。有在体研究表明,机械生长因子(MGF)可能能激活骨骼肌卫星细胞,并促进其增殖。本研究在此基础上通过离体实验,观察MGF对于骨骼肌卫星细胞的激活作用及促进其增殖的量效关系,探讨MGF对骨骼肌卫星细胞在细胞水平的调节机制,为骨骼肌损伤的修复提供了理论依据。 实验方法 原代骨骼肌卫星细胞取自4周龄雄性SD大鼠(2只,120g)双侧的腓肠肌与比目鱼肌。经0.1%Ⅱ型胶原酶结合0.25%胰蛋白酶消化法,通过两次差速贴壁纯化骨骼肌卫星细胞,使用横纹肌肌动蛋白鉴定骨骼肌卫星细胞的纯度。原代骨骼肌卫星细胞经两次传代后,使用不同浓度的MGF干预第三代细胞,浓度分别为10ng/ml,25ng/ml,50ng/ml, 100ng/ml,200ng/ml,在干预24h、48h、72h、96h后利用MTT法进行增殖效果的检测。使用25ng/ml的MGF对血清饥饿24h后的第三代骨骼肌卫星细胞进行干预,对比DMEM干预的骨骼肌卫星细胞,分别在8h、16h、24h、32h后使用流式细胞仪检测其所处细胞周期,测定其激活情况。 实验结果 1.骨骼肌卫星细胞活力达到98.8%。 2.骨骼肌卫星细胞纯度为97.4%。 3.MTT比色法的A值显示：48h：25ng／ml组、50ng／ml组A值显著高于对照组(P0.01), 10ng/ml组A值高于对照组(P0.05), 100ng/ml组、200ng/ml组与对照组之间差异没有显著性(P0.05), 100ng/ml与200ng/ml组的促增殖作用差异没有显著性(P0.05)；96h:10ng/ml与对照组之间差异没有显著性(P0.05),25ng/ml组、50ng/ml组、100ng/ml组、200ng/ml组A值显著高于对照组和10ng/ml组(P0.01),25ng/ml组、50ng/ml组、100ng/ml组、200ng/ml组之间差异没有显著性(P0.05)。低剂量的MGF即可促进骨骼肌卫星细胞的增殖,随浓度升高至25ng/ml至50ng/ml时,MGF促骨骼肌卫星细胞增殖作用相对更明显,浓度继续升高,会出现增殖速度减缓现象；各个时相检测后发现MGF干预到96h增殖速度减缓。结合时间-剂量的结果显示,在48h时25ng/ml组和50ng/ml组有交互作用。 4.使用不含血清的DMEM饥饿骨骼肌卫星细胞24h后,处于GO期的细胞达到了86.76% 5.25ng/ml MGF干预GO期的卫星细胞后,8h时MGF组和对照组之间差异没有显著性(P0.05),16h时MGF组G0／G 1期细胞含量低于对照组(P0.05),24h时MGF组低于对照组(P0.01),32h时MGF组低于对照组(P0.05)。 结论 1.MGF可以促进骨骼肌卫星细胞增殖。 2.MGF促进骨骼肌卫星细胞增殖呈现剂量和时间依赖性,较理想剂量范围在25ng/ml至50ng/ml之间,干预24h后即开始增殖。 3.不含血清的DMEM饥饿骨骼肌卫星细胞24h可以使其退出细胞周期。 4.MGF可以激活四周龄SD大鼠的GO期骨骼肌卫星细胞。
[Abstract]:Objective to study the role of skeletal muscle satellite cells in skeletal muscle growth, development, injury and repair. In vivo studies have shown that the mechanical growth factor (MGF) may activate skeletal muscle satellite cells and promote their proliferation. On the basis of in vitro experiments, the effects of MGF on the activation and proliferation of skeletal muscle satellite cells were observed, and the regulatory mechanism of MGF on skeletal muscle satellite cells at cell level was explored. It provides a theoretical basis for the repair of skeletal muscle injury. Methods the primary skeletal muscle satellite cells were obtained from the bilateral gastrocnemius and soleus muscles of 4 week old male SD rats (2 rats, 120g). Skeletal muscle satellite cells were purified by 0.1% collagenase and 0.25% trypsin digestion, and the purity of skeletal muscle satellite cells was identified by rhabdomyactin. After two passages of primary skeletal muscle satellite cells, different concentrations of MGF were used to interfere with the third generation cells. The concentrations were 10 ng / ml, 25 ng / ml, 100 ng / ml / ml, 200 ng / ml, respectively. After 96 hours, the proliferative effect was detected by MTT method. 25ng/ml MGF was used to intervene the third generation skeletal muscle satellite cells after 24 hours of serum starvation. Compared with those treated with DMEM, the cell cycle was measured by flow cytometry at 8 h, 16 h, 24 h and 32 h, respectively. Its activation was measured. Experimental results 1. The activity of skeletal muscle satellite cells was 98.8%. 2. The purity of skeletal muscle satellite cells was 97.4%. The A value of 3.MTT colorimetry showed that A value of 48h:25ng/ml group, 50ng/ml group was significantly higher than that of control group (P0.01), A value of 10ng/ml group was higher than that of control group (P0.05), 100ng/ml group, 100ng/ml group. There was no significant difference between 200ng/ml group and control group (P0.05), but there was no significant difference between 100ng/ml group and 200ng/ml group (P0.05). There was no significant difference between 96h:10ng/ml and control group (P0.05). The A value of 25ng/ml group, 50ng/ml group, 100ng/ml group and 200ng/ml group was significantly higher than that of control group and 10ng/ml group (P0.01), 25ng/ml group, 50ng/ml group, and so on. There was no significant difference between 100ng/ml group and 200ng/ml group (P0.05). Low dose of MGF could promote the proliferation of skeletal muscle satellite cells. When the concentration increased to 25ng/ml to 50ng/ml, the effect of MGF on the proliferation of skeletal muscle satellite cells was more obvious, and the increase of MGF concentration would slow down the proliferation of skeletal muscle satellite cells. It was found that the proliferation rate of MGF decreased at 96 h after each phase detection. The results of time-dose analysis showed that there was interaction between 25ng/ml group and 50ng/ml group at 48 h. 4. After using serum-free DMEM starved skeletal muscle satellite cells for 24 hours, the number of cells in GO phase reached 86.76% after intervention with GO phase satellite cells, and there was no significant difference between MGF group and control group at 8 h (P0.05). The cell content of G _ 0 / G _ 1 phase in MGF group was lower than that in control group at 16 h (P0.05), MGF group was lower than control group at 24 h (P0.01), and MGF group was lower than control group at 32 h (P0.05). Conclusion 1.MGF can promote the proliferation of skeletal muscle satellite cells. 2.MGF promoted the proliferation of skeletal muscle satellite cells in a dose-and time-dependent manner. The optimal dose range was between 25ng/ml and 50ng/ml, and the proliferation began after 24 hours of intervention. 3. DMEM starved skeletal muscle satellite cells without serum could withdraw from cell cycle for 24 h. 4.MGF can activate GO phase skeletal muscle satellite cells of four-week old SD rats.
【学位授予单位】：上海体育学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
[Abstract]:Objective to study the role of skeletal muscle satellite cells in skeletal muscle growth, development, injury and repair. In vivo studies have shown that the mechanical growth factor (MGF) may activate skeletal muscle satellite cells and promote their proliferation. On the basis of in vitro experiments, the effects of MGF on the activation and proliferation of skeletal muscle satellite cells were observed, and the regulatory mechanism of MGF on skeletal muscle satellite cells at cell level was explored. It provides a theoretical basis for the repair of skeletal muscle injury. Methods the primary skeletal muscle satellite cells were obtained from the bilateral gastrocnemius and soleus muscles of 4 week old male SD rats (2 rats, 120g). Skeletal muscle satellite cells were purified by 0.1% collagenase and 0.25% trypsin digestion, and the purity of skeletal muscle satellite cells was identified by rhabdomyactin. After two passages of primary skeletal muscle satellite cells, different concentrations of MGF were used to interfere with the third generation cells. The concentrations were 10 ng / ml, 25 ng / ml, 100 ng / ml / ml, 200 ng / ml, respectively. After 96 hours, the proliferative effect was detected by MTT method. 25ng/ml MGF was used to intervene the third generation skeletal muscle satellite cells after 24 hours of serum starvation. Compared with those treated with DMEM, the cell cycle was measured by flow cytometry at 8 h, 16 h, 24 h and 32 h, respectively. Its activation was measured. Experimental results 1. The activity of skeletal muscle satellite cells was 98.8%. 2. The purity of skeletal muscle satellite cells was 97.4%. The A value of 3.MTT colorimetry showed that A value of 48h:25ng/ml group, 50ng/ml group was significantly higher than that of control group (P0.01), A value of 10ng/ml group was higher than that of control group (P0.05), 100ng/ml group, 100ng/ml group. There was no significant difference between 200ng/ml group and control group (P0.05), but there was no significant difference between 100ng/ml group and 200ng/ml group (P0.05). There was no significant difference between 96h:10ng/ml and control group (P0.05). The A value of 25ng/ml group, 50ng/ml group, 100ng/ml group and 200ng/ml group was significantly higher than that of control group and 10ng/ml group (P0.01), 25ng/ml group, 50ng/ml group, and so on. There was no significant difference between 100ng/ml group and 200ng/ml group (P0.05). Low dose of MGF could promote the proliferation of skeletal muscle satellite cells. When the concentration increased to 25ng/ml to 50ng/ml, the effect of MGF on the proliferation of skeletal muscle satellite cells was more obvious, and the increase of MGF concentration would slow down the proliferation of skeletal muscle satellite cells. It was found that the proliferation rate of MGF decreased at 96 h after each phase detection. The results of time-dose analysis showed that there was interaction between 25ng/ml group and 50ng/ml group at 48 h. 4. After using serum-free DMEM starved skeletal muscle satellite cells for 24 hours, the number of cells in GO phase reached 86.76% after intervention with GO phase satellite cells, and there was no significant difference between MGF group and control group at 8 h (P0.05). The cell content of G _ 0 / G _ 1 phase in MGF group was lower than that in control group at 16 h (P0.05), MGF group was lower than control group at 24 h (P0.01), and MGF group was lower than control group at 32 h (P0.05). Conclusion 1.MGF can promote the proliferation of skeletal muscle satellite cells. 2.MGF promoted the proliferation of skeletal muscle satellite cells in a dose-and time-dependent manner. The optimal dose range was between 25ng/ml and 50ng/ml, and the proliferation began after 24 hours of intervention. 3. DMEM starved skeletal muscle satellite cells without serum could withdraw from cell cycle for 24 h. 4.MGF can activate GO phase skeletal muscle satellite cells of four-week old SD rats.
【学位授予单位】：上海体育学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
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