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  

Effects of Interference and Overexpression of BAMBI Gene on the Phenotype and Function of Porcine Ovarian Granulosa Cells

作　　者：孙义姗 杨飞[1,2] 黄涛 SUN Yishan;YANG Fei;HUANG Tao(College of Animal Science and Technolongy,Shihezi University,Shihezi 832000,China;Tecon Biological Co.,Ltd.,Urumqi 830001,China)

机构地区：[1]石河子大学动物科技学院,石河子832000 [2]天康生物股份有限公司,乌鲁木齐830001

出　　处：《中国畜牧兽医》2021年第2期563-573,共11页China Animal Husbandry & Veterinary Medicine

基　　金：教育部留学归国人员科研启动基金(20131092);兵团科技创新人才计划项目(2020CB018)。

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。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-βsignaling 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-βRⅡincreased significantly(P<0.05),so that the expression of SMAD2 and SMAD3 increased significantly(P<0.05).When overexpressing of BAMBI,TGF-βRⅡdecreased 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-βsignaling pathway.

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 

分 类 号：Q782[生物学—分子生物学]