MGF对骨骼肌卫星细胞激活的影响及与其增殖的量效关系
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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(P<0.01), 10ng/ml组A值高于对照组(P<0.05), 100ng/ml组、200ng/ml组与对照组之间差异没有显著性(P>0.05), 100ng/ml与200ng/ml组的促增殖作用差异没有显著性(P>0.05)；96h:10ng/ml与对照组之间差异没有显著性(P>0.05),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(P<0.01),25ng/ml组、50ng/ml组、100ng/ml组、200ng/ml组之间差异没有显著性(P>0.05)。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(P>0.05),16h时MGF组G0／G 1期细胞含量低于对照组(P>0.05),24h时MGF组低于对照组(P<0.01),32h时MGF组低于对照组(P<0.05)。
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。
OBJECTIVE
During myogenesis, skeletal muscle satellite cells play a key role in the process of repair and renewal of myofibers. Studies have shown that MGF appears to initiate muscle satellite cell activation and proliferation in vivo. And based on that, it has been observed the effects of MGF on activation and the dose-dependent manner on proliferation of SC in vitro which increasing the number available for local repair, as well as the mechanism at the levels of molecular.
METHODS
The primary passage SC were derived from gastrocnemius muscle and soleus muscle of the Sprague-Dawley rats (4 weeks of age, male).-The SC were digested with 0.1% CollagenaseⅡand 0.25% Trypsin, purified with means of differential attachment technique and identified withα-sarcometric actin.
The third passage SC were treated with MGF at five different doses:lOng/ml, 25ng/ml,50ng/ml, 100ng/ml,200ng/ml. The cell proliferation rate was measured by MTT cell proliferation assay after 24h,48h,72h,96h and the best dose of proliferation was determined. The third passage SC were serum starved for 24h without FBS and then were divided equally into experimental groups for different time points and one operated group treated with MGF plus one normal control group cultured with DMEM. The cell cycle was detected with Flow Cytometry after 8h, 16h,24h and 32h.
RESULTS
1. Assessment of cell viability showed that the live SC proportion was 98.8%.
2. The immunocytochemistry showed that the proportion of a-sarcometric actin positive SC was 97.4%.
3. The proliferation of SC was positively correlated with the MTT A value. 48h:The A values of the operated groups which treated with MGF of 25ng/ml and 50ng/ml were significantly higher than that of normal control group (P<0.01). The A value of 10ng/ml group was higher than that of normal control group (P<0.05). There was no significant difference between 100ng/ml group and normal control group (P>0.05) and the same effect was seen between 200ng/ml group and normal control group (P>0.05). There was no significant difference between 100ng/ml group and 200ng/ml group (P>0.05). 96h:The A values of operated groups of 25ng/ml.50ng/ml. 100ng/ml and 200ng/ml were significantly higher than that of 10ng/ml group as well as normal control group (P<0.01). There was no significant difference among the operated groups of 25ng/ml.50ng/ml, 100ng/ml and 200ng/ml (P>0.05).There was no significant difference between 10ng/ml group and normal control group (P>0.05). With the dose increasing, the proliferation peaked ranging from 25ng/ml to 50ng/ml and was inhibited thereafter. As time went by, the proliferation was inhibited at 96h with the treatment of MGF. Combining the time with the dose, it could be observed that there was an interaction effect between 25ng/ml group and 50ng/ml group at 48h.
4. The proportion of SC in GO phase was 86.76% by serum starvation with serum-free DMEM for 24 hours.
5. After 8h and 16h incubation there was no significant difference between the operated group and the normal control group (P>0.05). And there were significant differences between the operated group and the normal control group, the proportion of the SC in G0/G1 phase of operated group treated with 25ng/ml MGF was significantly lower than that in normal control group after 24h (P<0.01) as well as 32h (P<0.05).
CONCLUSIONS
1. MGF increases skeletal muscle SC proliferation.
2. MGF promotes skeletal muscle SC to proliferate in a dose-dependent and a time-dependent manner. And the ideal range of dose may be from 25ng/ml to 50ng/ml. SC begin to proliferate at 24h with the treatment of MGF.
Serum starvation without FBS can synchronize SC in GO phase within 24h.
3. MGF appears to initiate skeletal muscle SC activation of 4-week-old Sprague-Dawley rats.
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(P<0.01), 10ng/ml组A值高于对照组(P<0.05), 100ng/ml组、200ng/ml组与对照组之间差异没有显著性(P>0.05), 100ng/ml与200ng/ml组的促增殖作用差异没有显著性(P>0.05)；96h:10ng/ml与对照组之间差异没有显著性(P>0.05),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(P<0.01),25ng/ml组、50ng/ml组、100ng/ml组、200ng/ml组之间差异没有显著性(P>0.05)。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(P>0.05),16h时MGF组G0／G 1期细胞含量低于对照组(P>0.05),24h时MGF组低于对照组(P<0.01),32h时MGF组低于对照组(P<0.05)。
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。
OBJECTIVE
During myogenesis, skeletal muscle satellite cells play a key role in the process of repair and renewal of myofibers. Studies have shown that MGF appears to initiate muscle satellite cell activation and proliferation in vivo. And based on that, it has been observed the effects of MGF on activation and the dose-dependent manner on proliferation of SC in vitro which increasing the number available for local repair, as well as the mechanism at the levels of molecular.
METHODS
The primary passage SC were derived from gastrocnemius muscle and soleus muscle of the Sprague-Dawley rats (4 weeks of age, male).-The SC were digested with 0.1% CollagenaseⅡand 0.25% Trypsin, purified with means of differential attachment technique and identified withα-sarcometric actin.
The third passage SC were treated with MGF at five different doses:lOng/ml, 25ng/ml,50ng/ml, 100ng/ml,200ng/ml. The cell proliferation rate was measured by MTT cell proliferation assay after 24h,48h,72h,96h and the best dose of proliferation was determined. The third passage SC were serum starved for 24h without FBS and then were divided equally into experimental groups for different time points and one operated group treated with MGF plus one normal control group cultured with DMEM. The cell cycle was detected with Flow Cytometry after 8h, 16h,24h and 32h.
RESULTS
1. Assessment of cell viability showed that the live SC proportion was 98.8%.
2. The immunocytochemistry showed that the proportion of a-sarcometric actin positive SC was 97.4%.
3. The proliferation of SC was positively correlated with the MTT A value. 48h:The A values of the operated groups which treated with MGF of 25ng/ml and 50ng/ml were significantly higher than that of normal control group (P<0.01). The A value of 10ng/ml group was higher than that of normal control group (P<0.05). There was no significant difference between 100ng/ml group and normal control group (P>0.05) and the same effect was seen between 200ng/ml group and normal control group (P>0.05). There was no significant difference between 100ng/ml group and 200ng/ml group (P>0.05). 96h:The A values of operated groups of 25ng/ml.50ng/ml. 100ng/ml and 200ng/ml were significantly higher than that of 10ng/ml group as well as normal control group (P<0.01). There was no significant difference among the operated groups of 25ng/ml.50ng/ml, 100ng/ml and 200ng/ml (P>0.05).There was no significant difference between 10ng/ml group and normal control group (P>0.05). With the dose increasing, the proliferation peaked ranging from 25ng/ml to 50ng/ml and was inhibited thereafter. As time went by, the proliferation was inhibited at 96h with the treatment of MGF. Combining the time with the dose, it could be observed that there was an interaction effect between 25ng/ml group and 50ng/ml group at 48h.
4. The proportion of SC in GO phase was 86.76% by serum starvation with serum-free DMEM for 24 hours.
5. After 8h and 16h incubation there was no significant difference between the operated group and the normal control group (P>0.05). And there were significant differences between the operated group and the normal control group, the proportion of the SC in G0/G1 phase of operated group treated with 25ng/ml MGF was significantly lower than that in normal control group after 24h (P<0.01) as well as 32h (P<0.05).
CONCLUSIONS
1. MGF increases skeletal muscle SC proliferation.
2. MGF promotes skeletal muscle SC to proliferate in a dose-dependent and a time-dependent manner. And the ideal range of dose may be from 25ng/ml to 50ng/ml. SC begin to proliferate at 24h with the treatment of MGF.
Serum starvation without FBS can synchronize SC in GO phase within 24h.
3. MGF appears to initiate skeletal muscle SC activation of 4-week-old Sprague-Dawley rats.
引文
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