干扰OPN基因对贵州黑山羊卵巢颗粒细胞增殖、凋亡及繁殖相关基因的影响  

Effects of interference with OPN gene on proliferation,apoptosis and reproduction related genes of ovarian granulosa cells in Guizhou black goat

作　　者：马丝 夏薇 温晓艳 潘智仁 付业海 刘彬 陆情梅 赵佳福 MA Si;XIA Wei;WEN Xiaoyan;PAN Zhiren;FU Yehai;LIU Bing;LU Qingmei;ZHAO Jiafu(College of Animal Science,Guizhou University/Key Laboratory of Genetics,Breeding and Reproduction of Plateau Mountain Animals of Ministry of Education/Guizhou Province Key Laboratory of Animal Genetics,Breeding and Reproduction,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院/高原山地动物遗传育种与繁殖教育部重点实验室/贵州省动物遗传育种与繁殖重点实验室,贵州贵阳550025

出　　处：《畜牧与兽医》2025年第5期1-8,共8页Animal Husbandry & Veterinary Medicine

基　　金：大学生SRT计划项目(贵大SRT字[2022]085号);贵州省科技计划项目(黔科合支撑[2022]一般089);贵州省“生物育种先导性研究”项目(黔科合支撑[2022]重点033);贵州省科技创新团队项目(黔科合平台人才-CXTD[2023]025)。

摘　　要：旨在探究骨桥蛋白(OPN)基因与贵州黑山羊卵巢颗粒细胞(OGCs)增殖和凋亡间的关系。以贵州黑山羊为研究对象,通过构建OPN基因的4个干扰载体,转染至OGCs,并采用实时荧光定量PCR技术检测不同干扰载体对OPN基因的抑制效率,筛选最佳干扰载体;采用细胞计数试剂盒-8(CCK8)检测细胞增殖活力,细胞划痕试验检测其迁移能力;实时荧光定量PCR(RT-qPCR)检测与其相关的繁殖标记基因和凋亡相关基因的表达情况。结果:成功构建了OPN基因的4个干扰载体,与shRNA-NC组相比,shRNA-OPN-222、shRNA-OPN-385和shRNA-OPN-741均极显著抑制了OPN基因的表达(P<0.01),且shRNA-OPN-222抑制效果最佳;CCK8和细胞划痕试验结果表明,与shRNA-NC组相比,shRNA-OPN-222极显著抑制了OGCs的增殖活力和迁移能力(P<0.01);RT-qPCR结果发现沉默OPN基因极显著抑制了核心蛋白聚糖2(DCN2)、细胞色素P450家族19亚家族A肽1(CYP19A1)和雌激素受体1(ESR1)的基因表达(P<0.01),显著促进了β-连环蛋白结合因子2(CTNNB2)、胱天蛋白酶3(Caspase-3)和Bcl-2关联X蛋白(Bax)的基因表达(P<0.05),对铁蛋白重链1(FTH1)、胱天蛋白酶9(Caspase-9)的基因表达无显著影响。结论:干扰OPN基因,可以有效抑制OGCs的增殖和迁移,降低繁殖相关基因的表达水平,同时促进细胞的凋亡进程,这一研究为进一步阐明OPN基因影响山羊繁殖率的分子机制奠定了基础。The aim of this study was to investigate the relationship between the osteopontin(OPN) gene and proliferation and apoptosis of ovarian granulosa cells(OGCs) in Guizhou black goats.Using Guizhou black goat as the research object,we screened the interfering vectors by constructing four interfering vectors of OPN gene,transfecting them into OGCs,and detecting the inhibition efficiency of different interfering vectors on the OPN gene by real-time fluorescence quantitative PCR(RT-qPCR).We used cell counting kit-8(CCK8) to detect the proliferation viability of the cells,and the cell scratch assay to detect the migration ability.And we detected the expression of the reproductive marker genes and the apoptosis-related genes associated with them by fluorescence quantitative PCR.In this study,four interference vectors of the OPN gene were successfully constructed.shRNA-OPN-222,shRNA-OPN-385 and shRNA-OPN-741 all suppressed the gene expression of the OPN extremely significantly,compared with the shRNA-NC group(P<0.01),and the suppression effect of shRNA-OPN-222 was the most effective.The results of the CCK8 and cell scratch assay showed that shRNA-OPN-222 highly significantly inhibited the proliferative vigour and migration ability of OGCs,compared with the shRNA-NC group(P<0.01).The RT-qPCR results showed that si-lencing the OPN gene highly significantly inhibited the gene expression of the DCN2,CYP19A1 and ESR1(P<0.01),significantly promo-ted the gene expression of CTNNB2,Caspase-3 and Bax(P<0.05),and had no significant effect on the gene expression of the FTH1,Caspase-9.In conclusion,this study indicated that interference with the OPN gene effectively inhibited the proliferation and migration ofOGCs,lowered the expression level of the reproduction-related genes,and simultaneously promoted the apoptotic process of the cells.Theseresults laid a foundation for further elucidation of the molecular mechanism by which the OPN gene affects the reproductive rate of goats.

关 键 词：OPN基因 贵州黑山羊 卵巢颗粒细胞 细胞增殖 繁殖相关基因 

分 类 号：S814[农业科学—畜牧学]