干扰MSTN对绵羊成肌细胞增殖分化及相关基因表达的影响

doi:10.11843/j.issn.0366-6964.2018.01.006

Abstract

Abstract:

The purpose of this study was to explore the regulation mechanism of MSTN gene on the proliferation and differentiation of sheep myoblasts and further reveal the biological function of MSTN. The expression of endogenous MSTN gene of sheep myoblasts was interfered by recombinant adenovirus-mediated shRNA. CCK-8 assay was used to detect the proliferation of myoblasts, and flow cytometry analysis was used to detect cell cycle of myoblasts. The qRT-PCR was used to measure the mRNA expression of p21 in myoblasts. Immunocytochemistry was used to detect myotubes formation and the cell fusion rate was counted, qRT-PCR was used to measure the mRNA expression of the MyoD,MyoG,Myf5, Myf6 and HACD1 genes at 48, 72, 96 h after myoblast induced to differentiation. The results showed that the proliferation of myoblasts was inhibited after downregulating MSTN expression, the cell cycle was arrested at G0/G1 phase, and the expression of cell cycle inhibitor p21 increased. In the study on myoblast differentiation, we found the expression of 4 myogenic regulatory factors after inducing for 48 h were decreased, and the expression of MyoG gene decreased significantly(P<0.05);the myoblast fusion rate of interference group was significantly higher than that of negative control group(P<0.01) after inducing for 72 h, the expression of Myf5 and Myf6 genes decreased significantly (P<0.01), the expression of MyoD gene didn't increase significantly (P>0.05), the expression of MyoG gene increased significantly(P<0.01);the expression of Myf5(P<0.05), MyoD(P>0.05) and MyoG(P<0.01) decreased after inducing for 96 h, and the expression of Myf6 gene didn't increase significantly(P>0.05). In addition, the expression of HACD1 gene was significantly higher in the interference group than that in the negative control group (P<0.05) after inducing for 72 h, and no significant changes were detected after inducing for 48 or 96 h. The results show that the MSTN gene interfered could inhibit the proliferation while promote differentiation of myoblasts, and HACD1 gene is related to myoblast fusion.

CLC Number:

S826.2

WANG Hong-na, SUN Hong-xin, ZHANG Ying-jie, LIU Yue-qin, GU Zhen-hui, SHI Xiu-fen. Effects of Interfering MSTN on Proliferation and Differentiation of Sheep Myoblasts and Expression of Related Genes[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(1): 46-54.

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Effects of Interfering MSTN on Proliferation and Differentiation of Sheep Myoblasts and Expression of Related Genes

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