TGFβR1介导TGF-β/Smad信号通路对绵羊颗粒细胞功能的影响

doi:10.11843/j.issn.0366-6964.2023.08.019

摘要/Abstract

摘要： 旨在探究TGFβR1介导TGF-β/Smad信号通路调控绵羊颗粒细胞功能的机制。本试验收集绵羊卵巢颗粒细胞，通过构建pcDNA3.1（+）-TGFβR1和pcDNA3.1（+）-SMAD4过表达质粒载体及shRNA-TGFβR1和shRNA-SMAD4干扰表达质粒载体，对绵羊颗粒细胞中TGFβR1和SMAD4基因分别进行过表达和干扰，利用CCK-8细胞增殖检测、流式细胞术、Annexin-V FITC/PI双染技术检测绵羊颗粒细胞增殖、细胞周期及凋亡功能，并结合Real-Time PCR及Western blot检测TGFβR1、SMAD4的mRNA水平和细胞凋亡相关蛋白BAX、BCL2、Caspase3的表达水平及TGF-β/Smad信号通路中TGFβR1、TGFβR2、SMAD2/3的磷酸化水平。结果显示，过表达TGFβR1和SMAD4均极显著促进颗粒细胞从G0/G1期进入S期的能力（P<0.01）、颗粒细胞增殖及抗凋亡蛋白BCL2的表达（P<0.01），抑制凋亡蛋白BAX和Caspase3表达（P<0.01）。干扰TGFβR1和SMAD4均极显著促进颗粒细胞凋亡及凋亡蛋白BAX和Caspase3的表达（P<0.01），抑制抗凋亡蛋白BCL2的表达（P<0.01）。进一步研究发现，过表达TGFβR1可促进TGF-β/Smad信号通路中TGFβR1、TGFβR2及SMAD2/3磷酸化水平（P<0.01），并极显著促进SMAD4的蛋白表达水平（P<0.01）；干扰TGFβR1则显著抑制TGFβR1、TGFβR2（P<0.01）及SMAD2/3的磷酸化水平（P<0.05），并抑制SMAD4的蛋白表达（P<0.01）；过表达SMAD4则会显著促进TGFβR1蛋白的表达（P<0.01）。研究表明，TGFβR1促进绵羊颗粒细胞的增殖及周期进程，抑制颗粒细胞凋亡，通过介导TGF-β/Smad信号通路中SMAD2/3及SMAD4的表达正向调控绵羊颗粒细胞的增殖与周期，并负向调控其凋亡。

关键词: TGFβR1, 绵羊, 颗粒细胞, TGF-β/Smad信号通路

Abstract: The study aimed to explore the mechanism of the TGF-β/Smad signaling pathway mediated by TGFβR1 regulating the function of sheep granulosa cells. In this study, sheep ovarian granulosa cells were collected, and pcDNA3.1(+)-TGFβR1 and pcDNA3.1(+)-SMAD4 overexpressed plasmid vectors and shRNA-TGFβR1 and shRNA-SMAD4 interfering plasmid vectors were constructed to overexpress and interfere with TGFβR1 and SMAD4 genes in sheep granulosa cells, respectively. The proliferation, cell cycle and apoptosis of sheep granulosa cells were detected by CCK-8 cell proliferation assay, flow cytometry and Annexin-V FITC/PI double staining. The mRNA levels of TGFβR1 and SMAD4, the protein expression levels of apoptosis-related proteins BAX, BCL2, and Caspase3 and the phosphorylation levels of TGFβR1, TGFβR2 and SMAD2/3 in the TGF-β/Smad signaling pathway were detected by Real-Time PCR and Western blot. The results showed that overexpression of TGFβR1 and SMAD4 extremely significantly promoted the ability of granulosa cells to enter the S phase from the G0/G1 phase (P<0.01), the proliferation of granulosa cells and the expression of anti-apoptotic protein BCL2 (P<0.01), and inhibited the expression of apoptotic protein BAX and Caspase3 (P<0.01). Interference with TGFβR1 and SMAD4 both promoted granulosa cell apoptosis and the expressions of apoptotic protein BAX and Caspase3 (P<0.01) and inhibited the expression of anti-apoptotic protein BCL2 (P<0.01). Further studies showed that overexpression of TGFβR1 could promote the phosphorylation levels of TGFβR1, TGFβR2 and SMAD2/3 in the TGF-β/Smad signaling pathway (P<0.01), and extremely significantly promote the protein expression level of SMAD4 (P<0.01). Interference with TGFβR1 significantly inhibited the phosphorylation levels of TGFβR1, TGFβR2 (P<0.01) and SMAD2/3 (P<0.05), and inhibited the protein expression of SMAD4 (P<0.01). Overexpression of SMAD4 could significantly promote the expression of TGFβR1 protein (P<0.01). The results showed that TGFβR1 promoted the proliferation and cycle of sheep granulosa cells, inhibited granulosa cell apoptosis, positively regulated the proliferation and cycle of sheep granulosa cells, and negatively regulated the apoptosis of sheep granulosa cells by mediating the expression of SMAD2/3 and SMAD4 in TGF-β/Smad signaling pathway.

Key words: TGFβR1, sheep, granulosa cell, TGF-β/Smad signaling pathway

中图分类号:

S826.3

李悦欣, 刘爱菊, 马晓菲, 郑忠, 胡伯欣, 智云霞, 田树军. TGFβR1介导TGF-β/Smad信号通路对绵羊颗粒细胞功能的影响[J]. 畜牧兽医学报, 2023, 54(8): 3335-3347.

LI Yuexin, LIU Aiju, MA Xiaofei, ZHENG Zhong, HU Boxin, ZHI Yunxia, TIAN Shujun. Effect of TGFβR1 on the Function of Sheep Granulosa Cells Mediated by TGF-β/Smad Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3335-3347.

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