干扰MSTN对牛骨骼肌卫星细胞增殖分化的影响

doi:10.16431/j.cnki.1671-7236.2020.02.019

摘要/Abstract

摘要： 为研究肌肉生长抑制素（myostatin，MSTN）对牛骨骼肌卫星细胞增殖与成肌分化的影响，本试验以牛骨骼肌卫星细胞体外诱导成肌分化模型为对象，以前期设计合成3个干扰RNA（si-MSTN-1、si-MSTN-2、si-MSTN-3）并对其进行干扰效果筛选为基础，将干扰效果极显著的si-MSTN-2（si-MSTN）转染牛骨骼肌卫星细胞，通过EdU染色法检测干扰MSTN对牛骨骼肌卫星细胞增殖的影响；进一步对干扰MSTN的牛骨骼肌卫星细胞进行体外成肌诱导分化，通过肌管形成状态和分化标志因子综合分析干扰MSTN对牛骨骼肌卫星细胞分化的影响：首先通过显微镜观察牛骨骼肌卫星细胞分化时期的肌管形成状态，然后利用实时荧光定量PCR和Western blotting技术检测牛骨骼肌卫星细胞分化标志因子MyoG和MyHC在mRNA和蛋白水平的表达情况。结果显示，干扰MSTN后，牛骨骼肌卫星细胞中EdU阳性细胞率极显著增加（P < 0.01），说明下调MSTN表达极显著促进了牛骨骼肌卫星细胞的增殖；牛骨骼肌卫星细胞诱导分化后形成的肌管数量和直径均呈现增大趋势，牛骨骼肌卫星细胞成肌分化标志因子MyHC在mRNA和蛋白水平的表达均极显著高于对照组（P < 0.01），说明下调MSTN表达能够促进牛骨骼肌卫星细胞的成肌分化过程。本研究结果表明，干扰MSTN可以显著促进牛骨骼肌卫星细胞的增殖及成肌分化过程。本试验结果为进一步开展MSTN对牛骨骼肌卫星细胞成肌分化的调控机制研究提供了参考。

关键词: 肌肉生长抑制素(MSTN); 牛骨骼肌卫星细胞; 增殖; 成肌分化

Abstract: To explore the effect of myostatin (MSTN) on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells,the myogenic differentiation model induced by bovine skeletal muscle satellite cells in vitro was used in this study,three interfering RNAs (si-MSTN-1,si-MSTN-2 and si-MSTN-3) were designed and synthesized in the previous stage,si-MSTN-2(si-MSTN) with extremely significant interference effect was transfected into bovine skeletal muscle satellite cells to interfere with MSTN expression.The effect of interfering with MSTN on the proliferation of bovine skeletal muscle satellite cells was detected by EdU staining assay.In vitro myogenic differentiation of bovine skeletal muscle satellite cells transfected with si-MSTN was conducted,the effect of interfering with MSTN on myogenic differentiation of bovine skeletal muscle satellite cells was analyzed by myotube formation and the expression of differentiation markers.The myotube formation during the differentiation of bovine skeletal muscle satellite cells was observed by microscopy.The expression of skeletal muscle satellite cells differentiation markers MyoG and MyHC were detected using Real-time PCR and Western blotting at mRNA and protein levels.The results showed that EdU positive cell ratio in bovine skeletal muscle satellite cells extremely significantly increased after interfering with MSTN (P < 0.01).The number and diameter of myotubes formed in bovine skeletal muscle satellite cells after MSTN interference were extremely significantly higher than that of control group.At mRNA and protein levels,the expression of MyHC,a marker of myogenic differentiation,was extremely significantly increased after MSTN interference (P < 0.01).The results indicated that MSTN interference could significantly promote the proliferation and the process of myogenic differentiation of bovine skeletal muscle satellite cells promote.The results laid a foundation for further studies on the mechanism of MSTN on the myogenic differentiation of bovine skeletal muscle satellite cells.

Key words: MSTN; bovine skeletal muscle satellite cells; proliferation; myogenic differentiation

中图分类号:

S814.8

朱菲菲, 张俊星, 张林林, 李新, 刘新峰, 郭益文, 丁向彬. 干扰MSTN对牛骨骼肌卫星细胞增殖分化的影响[J]. 中国畜牧兽医, 2020, 47(2): 479-487.

ZHU Feifei, ZHANG Junxing, ZHANG Linlin, LI Xin, LIU Xinfeng, GUO Yiwen, DING Xiangbin. A Effect of Interfering with MSTN on the Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(2): 479-487.

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