干扰和过表达BAMBI基因对猪卵泡颗粒细胞表型和功能的影响

doi:10.16431/j.cnki.1671-7236.2021.02.018

摘要/Abstract

摘要： 为研究转化生长因子-β（TGF-β）信号通路中的骨形态发生蛋白和激活素膜结合抑制剂（bone morphogenetic protein and activin membrane-bound inhibitor，BAMBI）基因对猪卵泡颗粒细胞的调控作用，本研究设计构建BAMBI干扰和过表达载体，并将构建好的干扰（pSIREN-BAMBI-sh1、pSIREN-BAMBI-sh2、pSIREN-BAMBI-sh3）和过表达（pcDNA3.1-BAMBI）重组质粒转染猪卵泡颗粒细胞，通过实时荧光定量PCR技术进行有效片段的筛选，并对TGF-β信号通路下游基因（TGF-βRⅠ、TGF-βRⅡ、SMAD1、SMAD2、SMAD3、SMAD4、SMAD5）和细胞凋亡基因（Bax、Bcl-2）mRNA的表达水平进行检测。最后，用MTT法和流式细胞术检测BAMBI干扰和过表达对卵泡颗粒细胞增殖及凋亡的影响。结果表明，BAMBI干扰和过表达载体成功构建，pSIREN-BAMBI-sh2抑制BAMBI表达的效果最好，干扰效率最高。干扰BAMBI时，TGF-βRⅡ表达量显著上升（P<0.05），使得SMAD2、SMAD3的表达量显著上升（P<0.05）；过表达BAMBI时，TGF-βRⅡ表达显著下降（P<0.05），使得SMAD2、SMAD3的表达量显著下降（P<0.05）；上调BAMBI可显著抑制猪卵泡颗粒细胞增殖，极显著促进猪卵泡颗粒细胞凋亡（P<0.01）。研究结果表明，BAMBI基因显著影响猪卵泡颗粒细胞的生长发育，可能通过调节TGF-β信号通路间接影响猪的繁殖性能。

关键词: 猪; 转化生长因子-β(TGF-β); BAMBI基因; 颗粒细胞; 干扰; 过表达

Abstract: To investigate the regulation of bonemorphogenetic protein and activin membrane-bound inhibitor (BAMBI) gene in transforming growth factor-β (TGF-β) signaling pathway on porcine follicular granulosa cells,in this study,BAMBI interference vector and overexpression vector were designed and constructed.Porcine ovarian granulosa cells were ransfected with the constructed interference (pSIREN-BAMBI-sh1,pSIREN-BAMBI-sh2,pSIREN-BAMBI-sh3) and overexpression (pcDNA3.1-BAMBI) recombinant plasmids,and Real-time quantitative PCR technology was used to screen effective fragments and detect mRNA expression levels of TGF-β signaling pathway downstream genes (TGF-βRⅠ,TGF-βRⅡ,SMAD1,SMAD2,SMAD3,SMAD4,SMAD5) and apoptosis genes (Bax,Bcl-2).Finally,the effects of BAMBI interference and overexpression on the proliferation and apoptosis of ovarian granulosa cells were detected by MTT method and flow cytometry.The results showed that pSIREN-BAMBI-sh2 had the best effect of inhibiting BAMBI expression and the highest interference efficiency.When interfering with BAMBI,the expression of TGF-βRⅡ increased significantly (P<0.05),so that the expression of SMAD2 and SMAD3 increased significantly (P<0.05).When overexpressing of BAMBI,TGF-βRⅡ decreased significantly (P<0.05),so that the expression of SMAD2 and SMAD3 decreased significantly (P<0.05).Up-regulation of BAMBI inhibited the proliferation of granulosa cells and significantly promoted apoptosis (P<0.01).The results suggested that the BAMBI gene significantly affected the phenotype and function of ovarian granulosa cells,and might indirectly affect the reproductive performance of pigs by regulating the TGF-β signaling pathway.

Key words: pig; transforming growth factor-β (TGF-β); BAMBI gene; granulosa cells; interference; overexpression

中图分类号:

Q782

孙义姗, 杨飞, 黄涛. 干扰和过表达BAMBI基因对猪卵泡颗粒细胞表型和功能的影响[J]. 中国畜牧兽医, 2021, 48(2): 563-573.

SUN Yishan, YANG Fei, HUANG Tao. Effects of Interference and Overexpression of BAMBI Gene on the Phenotype and Function of Porcine Ovarian Granulosa Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 563-573.

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