PSMB5蛋白对牛骨骼肌卫星细胞增殖与成肌分化的影响

doi:10.3864/j.issn.0578-1752.2020.20.016

摘要/Abstract

摘要：

【目的】探究蛋白酶体β5亚基（proteasome subunit beta type-5, PSMB5）对牛骨骼肌卫星细胞增殖与成肌分化的影响,为进一步研究蛋白酶体亚基PSMB5在细胞分化和肌肉发育过程中的调控作用提供依据。【方法】利用牛骨骼肌卫星细胞体外诱导成肌分化模型,模拟牛骨骼肌生长发育过程,首先检测牛骨骼肌卫星细胞分化前后PSMB5的表达变化,采用qRT-PCR法检测PSMB5在牛骨骼肌卫星细胞分化前后基因表达水平的差异,采用Western blotting 法检测PSMB5在牛骨骼肌卫星细胞分化前后蛋白表达水平的差异。然后设计合成PSMB5的小干扰RNA si-RNA-PSMB5（si-PSMB5）,构建PSMB5过表达质粒载体pcDNA3.1-PSMB5（pcDNA-PSMB5）,采用（Lipofectamine）3000转染试剂将si-PSMB5和pcDNA-PSMB5转染牛骨骼肌卫星细胞,qRT-PCR法及Western blotting法检测转染效果。最后采用EdU染色的方法检测干扰和过表达PSMB5对牛骨骼肌卫星增殖的影响;进一步对牛骨骼肌卫星细胞进行体外成肌诱导分化,光学显微镜观察牛骨骼肌卫星细胞肌管形成状态,Western blotting检测分化标志因子肌球蛋白重链（MyHC）蛋白水平的表达变化,分析干扰和过表达PSMB5对牛骨骼肌卫星细胞成肌分化的影响,综合分析PSMB5蛋白对牛骨骼肌卫星细胞增殖与成肌分化的影响。【结果】PSMB5在牛骨骼肌卫星细胞分化前后表达水平存在显著差异,牛骨骼肌卫星细胞诱导分化后PSMB5的mRNA和蛋白表达量均显著高于增殖期（P<0.05）。干扰或过表达PSMB5后,牛骨骼肌卫星细胞EdU阳性细胞率与对照组相比,差异均不显著（P>0.05）。干扰PSMB5的表达后,细胞分化形成的肌管数量明显少于对照组,分化标志因子MyHC的蛋白表达水平显著低于对照组（P<0.05）;而过表达PSMB5后,细胞分化形成的肌管数量明显多于对照组,分化标志因子MyHC的蛋白表达水平显著高于对照组（P<0.05）。【结论】PSMB5蛋白对牛骨骼肌卫星细胞的增殖没有明显影响,但对牛骨骼肌卫星细胞的成肌分化过程具有显著的调控作用。干扰PSMB5表达可以抑制牛骨骼肌卫星细胞的成肌分化进程,而过表达PSMB5可以促进牛骨骼肌卫星细胞体外成肌分化进程。探明PSMB5蛋白对牛骨骼肌卫星细胞增殖和成肌分化的具体调控作用,为进一步开展PSMB5在牛成肌分化中的调控机制研究奠定了基础。

关键词: 牛, PSMB5, 骨骼肌卫星细胞, 细胞增殖, 成肌分化

Abstract:

【Objective】 The aim of this study was to investigate the effects of proteasome subunit beta type-5(PSMB5) on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells. 【Method】 In this study, the in vitro induced myogenic differentiation model of bovine skeletal muscle satellite cells was used to simulate the growth and development of bovine skeletal muscle. Firstly, the expression of PSMB5 in bovine skeletal muscle satellite cells before and after the differentiation was detected. The mRNA expression level of PSMB5 was detected by qRT-PCR, and the protein expression level of PSMB5 was detected by western blotting. Then, the small interfering RNA si-RNA-PSMB5 (si-PSMB5) was designed and synthesized, and PSMB5 overexpression plasmid pcDNA3.1-PSMB5 (pcDNA-PSMB5) was constructed, which were transfected into bovine skeletal muscle satellite cells by lipofectamine 3000, and the transfection effect was detected by qRT-PCR and Western blotting. Finally, EdU staining was used to detect the effects of PSMB5 interference and overexpression on the proliferation of bovine skeletal muscle satellite cells. Further, in vitro myogenic differentiation of bovine skeletal muscle satellite cells was induced, and the myotube formation of bovine skeletal muscle satellite cells was observed under light microscope. The expression of myosin heavy chain (MyHC) protein, a marker of differentiation, was detected by western blotting. The effects of PSMB5 on proliferation and differentiation of bovine skeletal muscle satellite cells were analyzed. 【Result】There was a significant difference in the expression level of PSMB5 before and after the differentiation of bovine skeletal muscle satellite cells. After the differentiation of bovine skeletal muscle satellite cells, the expression level of PSMB5 mRNA and protein was significantly higher than that in the proliferation period (P<0.05). When PSMB5 was interfered or overexpressed, there was no significant difference in EdU positive cell rate between the two groups (P>0.05). After interfering with the expression of PSMB5, the number of myotubes formed by cell differentiation was significantly less than that of the control group, and the protein expression level of MyHC was significantly lower than that of the control group (P<0.05); however, after overexpression of PSMB5, the number of myotubes formed by cell differentiation was significantly higher than that of the control group, and the protein expression level of MyHC was significantly higher than that of the control group (P<0.05). 【Conclusion】The results showed that the PSMB5 had no significant effect on the proliferation of bovine skeletal muscle satellite cells, but it had a significant regulatory effect on the myogenic differentiation process of bovine skeletal muscle satellite cells. Interference with PSMB5 expression could inhibit the myogenic differentiation process, while overexpression of PSMB5 could promote the myogenic differentiation process of bovine skeletal muscle satellite cells in vitro. In this study, the specific regulatory effect of PSMB5 on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells was explored, which laid a foundation for further study on the regulatory mechanism of PSMB5 in bovine muscle myogenic differentiation.

Key words: bovine, PSMB5, skeletal muscle satellite cells, cell proliferation, myogenic differentiation

来裕婷,朱菲菲,王轶敏,郭宏,张林林,李新,郭益文,丁向彬. PSMB5蛋白对牛骨骼肌卫星细胞增殖与成肌分化的影响[J]. 中国农业科学, 2020, 53(20): 4287-4296.

LAI YuTing,ZHU FeiFei,WANG YiMin,GUO Hong,ZHANG LinLin,LI Xin,GUO YiWen,DING XiangBin. Effects of PSMB5 on the Proliferation and Myogenic Differentiation of Skeletal Muscle Satellite Cells[J]. Scientia Agricultura Sinica, 2020, 53(20): 4287-4296.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2020.20.016

https://www.chinaagrisci.com/CN/Y2020/V53/I20/4287

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引物名称 Primer name	引物序列 Primer sequence	片段长度 Fragment length
PSMB5-F	5′-TCAAGATGTCAGCCACACCC -3′	112
PSMB5-R	5′-ACCACGCCTCCATACACAAG -3′	112
MyHC-F	5′- CTGGAATCCGGAGGCAGAA-3′	105
MyHC-R	5′- TTTTCGAAGGTAGGGAGCGG-3′	105
GAPDH-F	5′- TGTTGTGGATCTGACCTGCC-3′	135
GAPDH-R	5′- AAGTCGCAGGAGACAACCTG-3′	135