干扰核心蛋白聚糖对牛骨骼肌卫星细胞增殖分化的影响

doi:10.16431/j.cnki.1671-7236.2021.01.023

摘要/Abstract

摘要： 为探究肌肉生长抑制素（MSTN）对牛骨骼肌生长发育的作用机制，本研究以前期MSTN^+/-蒙古牛与野生蒙古牛腿臀肌肌肉组织定量蛋白质组学与磷酸化蛋白质组学筛选获得的表达差异倍数较大的核心蛋白聚糖（DCN）为靶标，以实验室前期分离培养的牛骨骼肌卫星细胞及建立的体外诱导成肌分化模型为对象，通过对设计合成的3个DCN siRNA干扰效果的筛选，将干扰效果最显著的si-DCN-2（si-DCN）转染牛骨骼肌卫星细胞。采用实时荧光定量PCR和Western blotting方法检测增殖期（GM）牛骨骼肌卫星细胞中增殖标志因子Pax7和MyoD的mRNA水平及蛋白水平的表达变化，以及使用EdU染色的方法检测干扰DCN对细胞增殖的影响。对转染DCN siRNA的牛骨骼肌卫星细胞进行体外成肌诱导分化，通过显微镜观察牛骨骼肌卫星细胞分化第3天（DM3）的肌管形成状态，同时采用实时荧光定量PCR和Western blotting检测分化标志因子MyoG和MyHC的mRNA水平及蛋白水平的表达变化，并对DM3期肌管MyHC进行免疫荧光染色，以研究干扰DCN对细胞分化的影响。结果显示，干扰DCN表达后，增殖期牛骨骼肌卫星细胞中Pax7和MyoD的mRNA水平及蛋白水平都显著或极显著上调（P<0.05；P<0.01），且EdU阳性细胞率显著增加（P<0.05），表明干扰DCN表达显著促进了牛骨骼肌卫星细胞的增殖。干扰DCN表达后，牛骨骼肌卫星细胞分化第3天诱导形成的肌管直径呈现增大趋势，检测成肌分化标志因子MyoG在mRNA和蛋白水平的表达分别极显著和显著高于对照组（P<0.01；P<0.05），MyHC在mRNA水平显著降低（P<0.05），但在蛋白水平上极显著升高（P<0.01），免疫荧光结果显示，下调DCN后肌管融合指数显著高于对照组（P<0.05），说明干扰DCN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DCN可以显著促进牛骨骼肌卫星细胞的增殖和成肌分化过程。研究结果为进一步开展MSTN对牛骨骼肌卫星细胞成肌分化的调控机制研究奠定了基础。

关键词: 核心蛋白聚糖(DCN); 牛; 骨骼肌卫星细胞; 增殖; 成肌分化

Abstract: In order to explore the mechanism of myostatin (MSTN) on the growth and development of bovine skeletal muscle,this study aimed at the expression of decorin (DCN) with large fold difference obtained by quantitative proteomic and phosphoproteomic screening of muscle tissues of MSTN^+/- Mongolian cattle and wild Mongolian calf gluteal muscle,and isolated and cultured bovine skeletal muscle satellite cells in the pre-laboratory.The model of induced myogenic differentiation in vitro was established as the object.Through screening the interference effects of three DCN siRNAs designed and synthesized,the most significant interference effect of si-DCN-2 (si-DCN) was transfected into bovine skeletal muscle satellite cells.Real-time fluorescence quantitative PCR and Western blotting were used to detect the changes in the expression of the proliferation markers Pax7 and MyoD in proliferative phase (GM) bovine skeletal muscle satellite cells and the effects of interfering DCN on cell proliferation was detected using EdU staining.In vitro myogenic differentiation of bovine skeletal muscle satellite cells transfected with DCN siRNA was performed.Myotube formation on the third day of differentiation (DM3) of bovine skeletal muscle satellite cells was observed by microscopy,and Real-time fluorescence quantitative PCR and Western blotting were used to detecte the expression changes of the differentiation markers MyoG and MyHC at the mRNA level and protein level,and myotube MyHC at DM3 stage was stained by immunofluorescence to study the effect of interfering with DCN on cell differentiation.The results showed that after interfering with DCN expression,the mRNA and protein levels of Pax7 and MyoD in bovine skeletal muscle satellite cells in the proliferative phase were significantly or extremely significantly upregulated (P<0.05;P<0.01),and the rate of EdU-positive cells was significantly increased (P<0.05),indicating that interfering with DCN expression significantly promoted the proliferation of bovine skeletal muscle satellite cells.After interfering with the expression of DCN,the diameter of myotubes induced by bovine skeletal muscle satellite cells on the third day of differentiation showed an increasing trend.The expression of MyoG,a marker of myogenic differentiation,was extremely significantly and significantly higher than that of the control group at the mRNA and protein levels respectively (P<0.01;P<0.05).MyHC was significantly lower at the mRNA level (P<0.05),but significantly higher at the protein level (P<0.01).Immunofluorescence results showed that the myotube fusion index was significantly higher after downregulation of DCN than that of the control group (P<0.05),indicating that interfering with DCN expression can promote the myogenic differentiation process of bovine skeletal muscle satellite cells.The results of this study showed that interfering with DCN could significantly promote the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells.The results laid a foundation for further research on the regulatory mechanism of MSTN on myogenic differentiation of bovine skeletal muscle satellite cells.

Key words: decorin (DCN); bovine; skeletal muscle satellite cells; proliferation; myogenic differentiation

中图分类号:

S813.3

盛辉, 郭益文, 张林林, 苗曼宁, 李新, 丁向彬, 郭宏. 干扰核心蛋白聚糖对牛骨骼肌卫星细胞增殖分化的影响[J]. 中国畜牧兽医, 2021, 48(1): 209-217.

SHENG Hui, GUO Yiwen, ZHANG Linlin, MIAO Manning, LI Xin, DING Xiangbin, GUO Hong. Effects of Interfering with Decorin on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 209-217.

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