BMP4/SMAD4通过下调GJA 1基因表达影响绵羊卵巢颗粒间隙连接活性  

BMP4/SMAD4 Downregulates GJA 1 Gene Expression to Affect the Gap Junctional Intercellular Communication Activity in Sheep Ovarian Granulosa Cells

作　　者：何雨 王翔宇[2] 狄冉[2] 储明星[2] 梁琛[1] HE Yu;WANG Xiangyu;DI Ran;CHU Mingxing;LIANG Chen(College of Animal Science,Shanxi Agricultural University,Taigu 030801,China;State Key Laboratory of Animal Biotech Breeding,Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]山西农业大学动物科学学院,太谷030801 [2]中国农业科学院北京畜牧兽医研究所,畜禽生物育种全国重点实验室,北京100193

出　　处：《畜牧兽医学报》2025年第2期679-688,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：中央级公益性科研院所基本科研业务费专项(Y2024YJ08);财政部和农业农村部国家现代农业产业技术体系资助(CARS-38);山西省基础研究计划面上项目(20210302123371);山西农业大学博士科研启动项目(2021BQ04)。

摘　　要：旨在探索骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)对绵羊卵巢颗粒细胞中间隙连接蛋白基因(gap junction protein alpha 1,GJA 1)表达的影响及其分子调控机制。本研究利用廊坊市屠宰场收集的2~4岁健康绵羊卵巢分离颗粒细胞,采用免疫荧光染色技术定位GJA1在颗粒细胞中的分布。将细胞随机分为4组,分别添加0、10、50和100 ng·mL^(-1)浓度的重组BMP4蛋白,每组3个重复,培养24 h,利用CCK-8法评估细胞活性,RT-qPCR和Western blot研究BMP4对GJA 1的mRNA和蛋白表达水平的影响。为探究BMP4调控GJA 1表达的潜在机制,将细胞随机分为3组,每组3个重复,除对照组外分别添加10μmol·L^(-1)BMP I型受体抑制剂(Dorsomorphin)和干扰小RNA敲除SMAD家族蛋白4(SMAD family member 4,SMAD 4),均添加100 ng·mL^(-1)的BMP4处理细胞24 h,RT-qPCR检测GJA 1和SMAD 4表达量,Western blot分析测定GJA1和SMAD4表达水平以及SMAD1/5/8的磷酸化水平,最后利用划痕染料示踪试验检测绵羊卵巢颗粒细胞之间的间隙连接活性。结果显示,BMP4显著抑制了绵羊卵巢颗粒细胞中GJA 1表达和间隙连接活性(P<0.05),此抑制效应在添加Dorsomorphin和敲除SMAD 4后显著减弱(P<0.05),同时,BMP4处理显著增加了SMAD1/5/8的磷酸化水平(P<0.05)。综上,BMP4通过SMAD1/5/8-SMAD4信号转导调控GJA 1表达进而影响颗粒细胞间隙连接活性,本结果增加了对绵羊BMP/SMAD通路调控颗粒细胞间隙连接活性的了解,为改进体外卵泡成熟方法和高繁母羊的分子育种提供了基础。This study aimed to explore the effect of bone morphogenetic protein 4(BMP4)on the expression of gap junction protein alpha 1(GJA 1)in sheep ovarian granulosa cells and its molecular regulatory mechanism.Granulosa cells were isolated from the ovaries of healthy 2-4-year-old sheep obtained from a slaughterhouse in Langfang city.Immunofluorescence staining was used to localize the distribution of GJA1 in granulosa cells.The cells were randomly divided into 4 groups,each treated with recombinant BMP4 protein at concentrations of 0,10,50,and 100 ng·mL^(-1),with 3 replicates per group,and cultured for 24 hours.Cell viability was assessed using the CCK-8 assay,while RT-qPCR and Western blot were employed to examine the effects of BMP4 on GJA 1 mRNA and protein expression levels.To investigate the potential mechanism by which BMP4 regulates GJA 1 expression,the cells were divided into 3 groups with 3 replicates per group.In addition to the control group,cells were treated with 10μmol·L^(-1)BMP type I receptor inhibitor(Dorsomorphin)and small interfering RNA to knock down SMAD family member 4(SMAD 4),all treated with 100 ng·mL^(-1)BMP4 for 24 hours.RT-qPCR was used to measure the expression of GJA 1 and SMAD 4,and Western blot was used to assess the expression levels of GJA1 and SMAD4 as well as the phosphorylation levels of SMAD1/5/8.Finally,a scratch dye tracking assay was performed to assess the gap junction activity between sheep ovarian granulosa cells.The results showed that BMP4 significantly inhibited the expression of GJA 1 and gap junction activity in sheep ovarian granulosa cells(P<0.05).This inhibitory effect was significantly weakened after treatment with Dorsomorphin and SMAD 4 knockdown(P<0.05).Moreover,BMP4 treatment significantly increased the phosphorylation levels of SMAD1/5/8(P<0.05).In conclusion,BMP4 regulates GJA 1 expression and subsequently affects the gap junction activity of granulosa cells through the SMAD1/5/8-SMAD4 signaling pathway.These findings enhance the understanding of how the BMP

关 键 词：绵羊 卵巢颗粒细胞 BMP4 间隙连接 GJA1 SMAD 4 

分 类 号：S826.3[农业科学—畜牧学]