目的 探讨新生个体骨骼肌生长重要营养因子亮氨酸对新生大鼠骨骼肌卫星细胞肌管形成和肌球蛋白重链(myosin heavy chain,MyHC)表达影响的机制。方法 分离纯化原代新生大鼠骨骼肌卫星细胞,在细胞的分化阶段用不同浓度亮氨酸和雷帕霉素(0 mM亮氨酸,0.1 mM亮氨酸,0.5 mM亮氨酸,2.0 mM亮氨酸,2.0 mM亮氨酸+50 nM雷帕霉素)处理细胞,观察肌管形成情况并检测MyHC、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、p-mTOR和Myod的表达。结果 亮氨酸促进肌管的形成,随着原代新生大鼠骨骼肌卫星细胞分化MyHC表达逐渐增加(P<0.05、<0.01或<0.001)。亮氨酸浓度为0.5 mM和2.0 mM时原代新生大鼠骨骼肌卫星细胞p-mTOR表达水平高于亮氨酸浓度为0 mM组(P<0.05或<0.01),添加雷帕霉素后,p-mTOR表达水平下降(P<0.05)。亮氨酸浓度为0.5 mM和2.0 mM时原代新生大鼠骨骼肌卫星细胞Myod的表达水平高于亮氨酸浓度为0 mM组(P值均<0.05),雷帕霉素则抑制Myod的表达(P<0.05)。亮氨酸浓度为0.5 mM和2.0 mM时原代新生大鼠骨骼肌卫星细胞MyHC的表达水平高于亮氨酸浓度为0 mM组(P<0.05或<0.01),雷帕霉素则抑制MyHC的表达(P<0.05)。结论 亮氨酸通过mTORC1/Myod信号通路上调新生大鼠骨骼肌卫星细胞MyHC表达,促进肌管的形成。
Abstract
Objective To explore the effect of leucine,the important nutrient for neonatal skeletal muscle growth on myotubes formation and myosin heavy chain (MyHC) expression of primary neonatal rat skeletal muscle satellite cells. Methods The primary neonatal rat skeletal muscle satellite cells were disassociated and purified.In the stage of cell differentiation,they were treated with different concentration of leucine and rapamycin ( 0mM leucine,0.1 mM leucine,0.5 mM leucine,2.0 mM leucine,2.0 mM leucine+50 nM rapamycin).Myotubes were observed and Western blot was performed to detect the expression of MyHC,mammalian target of rapamycin (mTOR),p-mTOR and MyoD. Results Leucine promoted the formation of myotubes.The expression of MyHC gradually increased with the differentiation of primary neonatal rat skeletal muscle satellite cells (P<0.05,P<0.01 or P<0.001).The expression of p-mTOR were higher in the 0.5 mM leucine group and 2.0 mM leucine group than that in the 0mM leucine group (P<0.05 or P<0.01).The expressions of p-mTOR were decreased by rapamycin (P<0.05).The expression of MyoD was higher in the 0.5 mM leucine group and 2.0 mM leucine group than that in the 0 mM leucine group (P<0.05).The expression of MyoD was decreased by rapamycin (P<0.05).The expression of MyHC were higher in the 0.5 mM leucine group and 2.0 mM leucine group than that in the 0 mM leucine group (P<0.05 or P<0.01).The expression of MyHC was decreased by rapamycin (P<0.05). Conclusion Leucine promotes the myotubes formation and MyHC expression of primary neonatal rat skeletal muscle satellite cells in part via mTORC1/MyoD signaling pathway.
关键词
亮氨酸 /
新生 /
骨骼肌卫星细胞 /
肌球蛋白重链 /
哺乳动物雷帕霉素靶蛋白
Key words
leucine /
neonate /
skeletal muscle satellite cell /
myosin heavy chain /
mammalian target of rapamycin
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基金
广东省自然科学基金(2015A030313148);广东省科技计划项目(2012B061700069);广东省大学生创新训练计划项目(201501143)
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