敲除G0S2基因对绵羊卵巢颗粒细胞增殖、类固醇激素及相关基因表达的影响  

Effects of Knockout of G0S2 Gene in Ovarian Granulosa Cell Proliferation,Steroids Hormones and Related Gene Expression

作　　者：马钰静 段春辉[1] 贺名扬 张英杰[1] 杨若晨 王泳[1] 刘月琴[1] MA Yu-jing;DUAN Chun-hui;HE Ming-yang;ZHANG Ying-jie;YANG Ruo-chen;WANG Yong;LIU Yue-qin(College of Animal Science and Technology,Agricultural University of Hebei,Baoding 071000)

机构地区：[1]河北农业大学动物科技学院,保定071000

出　　处：《生物技术通报》2023年第6期325-334,共10页Biotechnology Bulletin

基　　金：国家绒毛用羊产业技术体系(CARS-39);国家肉羊产业技术体系(CARS-38)。

摘　　要：本文旨在探究G0S2基因对绵羊卵巢颗粒细胞增殖,雌激素(E_(2))和孕酮(P_(4))分泌,以及类固醇分泌相关基因和细胞凋亡基因表达的影响。采集小尾寒羊新鲜卵巢,用割吸法收集中小卵泡的卵泡液,离心分离颗粒细胞,分为试验组和对照组,试验组采用CRISPR-Cas9基因编辑技术敲除G0S2基因,获得重组表达载体PX458-sgRNA-G0S2转染至颗粒细胞;对照组转染PX458质粒至颗粒细胞。检测颗粒细胞活力和凋亡情况,以及E_(2)和P_(4)水平及相关基因的表达量。结果表明,试验组G0S2 mRNA的表达量及蛋白水平极显著低于对照组(P<0.01),分别降低了66%和70%,G0S2敲除成功。试验组颗粒细胞的增殖活力显著高于对照组(P<0.05),细胞凋亡率极显著下降56%(P<0.01)。试验组E_(2)水平显著高于对照组(P<0.05),P_(4)水平显著低于对照组(P<0.05)。试验组颗粒细胞中StAR、CYP11、3β-HSD的表达量极显著低于对照组(P<0.01),CYP19的表达量极显著高于对照组(P<0.01)。与对照组相比,试验组颗粒细胞中Caspase3和Bax的表达极显著下调(P<0.01),Bcl-2的表达极显著上调(P<0.01)。上述结果表明,敲除G0S2基因能促进在绵羊卵巢颗粒细胞的增殖,抑制其凋亡,通过下调StAR、CYP11、3β-HSD,上调CYP19的表达影响E_(2)和P_(4)的分泌。The objective of this research is to explore the effects of the G0S2 gene on the cell proliferation of ovarian granulosa cells,the secretion of estrogen(E_(2))and progesterone(P_(4)),as well as the expressions of steroid secretion-related genes and apoptosis genes.Fresh ovaries of small-tailed sheep were collected,and the follicular fluid gathered from small and medium-sized follicles by the suction method was centrifuged to separate the granulosa cells into experimental and control groups.The G0S2 gene in the experimental group was knocked out by CRISPR-Cas9 gene editing technology and the obtained recombinant expression vector PX458-sgRNA-G0S2 was transfected to the granulosa cells.The PX458 plasmid in the control group was transfected to the granulosa cells.The viability and apoptosis of the granulosa cells were detected,as well as the E_(2) and P_(4) concentrations and associated gene expression.The results showed that the relative abundance and the protein level of G0S2 mRNA in the experimental group were lower(P<0.01)than those in the control group,reduced by 66%and 70%respectively,proving that the G0S2 was successfully knocked out.The proliferation activity of the granulosa cells in the experimental group was highe(r P<0.05)than the control group,and the apoptosis rate reduced(P<0.01)by 56%.Compared with the control group,the E_(2) concentration was higher(P<0.05)and the P_(4) concentration was lower(P<0.05)in the experimental group.The expressions of genes StAR,CYP11 and 3β-HSD in the experimental group was lower(P<0.01)than the control group,and the expressions of CYP19 was significantly higher(P<0.01)than the control group.Compared with the control group,the expression of Caspase3 and Bax in the experimental group of granulosa cells was significantly downregulated(P<0.01),and the expressions of Bcl-2 was significantly upregulated(P<0.01).Our results demonstrate that knockout of the G0S2 gene can promote the proliferation of ovarian granulosa cells,inhibit their apoptosis,and affect the secretion of E_(

关 键 词：G0S2 绵羊 颗粒细胞 增殖 凋亡 类固醇激素 

分 类 号：S826.2[农业科学—畜牧学]