TGF-β调节仔猪睾丸支持细胞增殖的机制

doi:10.11843/j.issn.0366-6964.2018.04.006

摘要/Abstract

摘要：

旨在研究TGF-β对仔猪睾丸支持细胞增殖相关蛋白及基因表达的影响，进一步揭示TGF-β调控仔猪支持细胞增殖的作用机制。本研究用TGF-β及TGF-β/Smad通路抑制剂LY2109761处理培养的仔猪睾丸支持细胞，通过CCK-8与流式细胞术检测支持细胞活性和细胞周期，Western blotting检测Smad3的蛋白磷酸化水平，实时定量PCR（RT-qPCR）检测c-Myc mRNA、Skp2 mRNA及ssc-miRNA-24的表达。此外，支持细胞转染ssc-miRNA-24模拟物、抑制剂后，用TGF-β处理支持细胞，检测相关基因和蛋白的表达。结果表明，TGF-β以剂量依赖方式影响支持细胞活性；180 pg·mL^-1 TGF-β以时间（0~24 h）依赖方式影响细胞增殖指数（Proliferation index，PI）和S期细胞比例（S-phase cell fraction，SPF），抑制Smad3磷酸化，增加c-Myc mRNA、Skp2 mRNA、ssc-miRNA-24的表达；10 μmol·L^-1 LY2109761促进TGF-β诱导支持细胞活性，并增加细胞增殖指数和S期细胞比例。ssc-miRNA-24模拟物抑制Smad3磷酸化，促进c-Myc、Skp2 mRNA表达；ssc-miRNA-24抑制剂则具有相反的效果。综上表明，在仔猪睾丸支持细胞中，TGF-β可通过增加ssc-miRNA-24的表达，抑制Smad3磷酸化，增强c-Myc与Skp2 mRNA的表达，促进支持细胞增殖。

Abstract:

The objective of the study was to investigate the effect of TGF-β on the expression of proliferation-related proteins and genes of boar Sertoli cells, and reveal the mechanism of TGF-β regulating proliferation of boar Sertoli cells. In this study, TGF-β and TGF-β/Smad pathway inhibitor LY2109761 was used to stimulate cultured immature boar Sertoli cells. Cell viability and cell cycle were detected by CCK-8 kit and flow cytometry, respectively. The phosphorylation levels of Smad3 were detected by Western blotting. The expressions of c-Myc mRNA, Skp2 mRNA and ssc-miRNA-24 were detected by real-time quantitative PCR (RT-qPCR). Furthermore, ssc-miRNA-24 mimics and inhibitor were transfected into Sertoli cells, and then Sertoli cells were treated with TGF-β, following detected the expression of related genes and proteins. The results showed that TGF-β influenced cell viability in a dose-dependent manner. 180 pg·mL^-1 TGF-β increased cell proliferation index (PI)and S-phase cell fraction (SPF), inhibited the phosphorylation of Smad3 in a time-dependent (0-24 h) manner, increased the abundance of c-Myc mRNA, Skp2 mRNA, ssc-miRNA-24. 10 μmol·L^-1 LY2109761 promoted TGF-β-induced cell viability, increased cell proliferation index and S-phase cell fraction. In addition, ssc-miRNA-24 mimics decreased phosphorylation level of Smad3, promoted the expression of c-Myc and Skp2 mRNA. However, ssc-miRNA-24 inhibitor had an opposite effect on these parameters. The above results revealed that TGF-β decreased phosphorylation level of Smad3 by increasing the expression of ssc-miRNA-24, and enhanced the expression of c-Myc and Skp2 mRNA to promote the proliferation of boar Sertoli cells.

中图分类号:

S828.2

黄莎, 孙思, 邓洁, 胡煜, 练雨, 王鲜忠. TGF-β调节仔猪睾丸支持细胞增殖的机制[J]. 畜牧兽医学报, 2018, 49(4): 701-708.

HUANG Sha, SUN Si, DENG Jie, HU Yu, LIAN Yu, WANG Xian-zhong. Mechanism of TGF-Beta Regulating Proliferation of Boar Sertoli Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 701-708.

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https://www.xmsyxb.com/CN/Y2018/V49/I4/701

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