斜带石斑鱼mstn对其肌肉细胞增殖分化及相关基因表达的影响

Effects of mstn on proliferation and differentiation of muscle cells and the expression of related genes in Epinephelus coioides

斜带石斑鱼Epinephelus coioides是具有较高经济价值的海水鱼,如何促进石斑鱼生产性状改良一直是科学研究的重点之一。肌肉生长抑制素(mstn,myostatin)是调节肌肉生长的重要转化因子。本研究中,Mstn的亚细胞定位表明Mstn在斜带石斑鱼细胞质中呈点状分布。利用酶标仪测定被Mstn重组蛋白刺激肌肉原代细胞1~5 d后的A值,发现实验组细胞生长明显减缓,随着处理时间的增加,细胞活性差异进一步扩大。利用流式细胞仪测定被Mstn重组蛋白刺激的肌肉原代细胞细胞周期,发现实验组细胞周期受阻。用Mstn重组蛋白对肌肉原代细胞进行刺激,通过RT-qPCR检测Mstn重组蛋白对mstn信号通路及下游相关基因的影响,结果表明随着处理浓度的升高,p21、smad3和mrf4 mRNA的表达量也逐渐而上升,其中,smad3和mrf4表达量的上升不存在显著性差异;当处理浓度为100和1000 nmol/L时,p21存在显著性差异;随着处理浓度的升高,myod和myog mRNA的表达量均下降,但myod mRNA下降趋势不显著,myog mRNA显著下降,当处理浓度为100和1000 nmol/L时存在显著性差异。本研究揭示了Mstn定位于斜带石斑鱼GS细胞质;Mstn重组蛋白上调smad3、mrf4的表达,下调myod、myog的表达,来促进细胞的分化;同时Mstn重组蛋白能上调p21的表达,抑制细胞的增殖。本研究验证了Mstn在肌肉发育过程中的生物学功能,为后续深入开展mstn对鱼类肌肉发育的影响研究奠定了基础。

Epinephelus coioides is a marine fish with high economic value.How to improve the production characteristics of E.coioides has always been one of the focuses of scientific research.Myostatin(mstn)is an important transforming factor in regulating muscle growth.In this study,we investigated the effects of mstn on proliferation and differentiation of muscle cells and its related genes expression.The subcellular localization of Mstn showed that Mstn distributed in the cytoplasm of E.coioides.The A value of muscle primary cells stimulated by Mstn recombinant protein for 1-5 days was measured by enzyme labeling instrument.It was found that the growth of cells in the experimental group slowed down significantly,and the difference of cell activity further expanded with the increase of treatment time.Primary muscle cells were stimulated with Mstn recombinant protein.The effects of Mstn recombinant protein on mstn signal pathway and downstream related genes were detected by RT-qPCR.The results showed that the expression of p21,smad3 and mrf4 mRNA increased gradually with the increase of treatment concentration,and there was no significant difference in the expression of smad3 and mrf4;When the treatment concentrations were 100 and 1000 nmol/L,there was a significant difference in p21;With the increase of treatment concentration,the expression of myod and myog mRNA decreased,but the downward trend of myod mRNA was not significant.Myog mRNA decreased significantly,and there was a significant difference when the treatment concentration was 100 and 1000 nmol/L.This study revealed that Mstn is localized in the cytoplasm of E.coioides GS;the recombinant Mstn protein up-regulates the expression of smad3 and mrf4,and down-regulates the expression of myod and myog to promote cell differentiation;at the same time,the recombinant Mstn protein can up-regulate the expression of p21 and inhibit the cell proliferation.The study verified the biological function of Mstn in the process of muscle development,and provided a theoretical foundation for the subsequent in-depth study of the effect of mstn on fish muscle development.