WNT2在绵羊卵泡颗粒细胞的表达及功能研究

Expression and Function Analysis of WNT2 in Ovine Follicular Granulosa Cells

旨在探究WNT2在绵羊卵泡颗粒细胞(GCs)中的表达及功能。本研究选取4~6月龄健康母羊20只,采集双侧卵巢,免疫组化技术检测WNT2蛋白在卵泡中的表达定位;qRT-PCR及Western blot技术检测其在不同发育阶段卵泡颗粒细胞中的表达差异;siRNA沉默GCs中的Wnt2基因后,qRT-PCR技术检测Wnt2基因及参与经典WNT信号通路关键基因CTNNB1的相对表达量,并测定GCs凋亡情况。结果表明:1)WNT2蛋白在绵羊卵泡内膜细胞、颗粒细胞以及卵丘细胞内均有表达。2)qRT-PCR及Western blot结果基本一致,均表明Wnt2 mRNA及蛋白在不同发育阶段卵泡颗粒细胞表达差异显著(P<0.05),且在大卵泡颗粒细胞内表达量显著高于中卵泡颗粒细胞(P<0.05),中卵泡颗粒细胞内表达量显著高于小卵泡颗粒细胞(P<0.05)。3)基因沉默后,沉默组Wnt2和CTNNB1的表达量均显著低于无义序列siRNA组(NC组)以及空白对照组(P<0.05),而Wnt2基因沉默组细胞凋亡率显著高于其他两组(P<0.05)。综上表明,WNT2是通过WNT2/CTNNB1信号通路促进绵羊卵泡颗粒细胞生物学功能的。

This study aimed to investigate the expression and function of WNT2 in ovine follicular granulosa cells(GCs).In this study,both ovaries of 20 healthy ewes aged 4-6 months were collected and the expression of WNT2 protein in follicles was localized by immunohistochemistry;qRT-PCR and Western blot were used to detect the differences of its expression in follicular granulosa cells at different developmental stages;The Wnt2 expression was silenced in GCs,and the relative expression of Wnt2 gene and the key gene CTNNB1 involved in the classical WNT signaling pathway were analyzed using qRT-PCR technology,and the apoptosis of GCs was detected.The results showed that:1)WNT2 protein was expressed in theca interna cells,granulosa cells and cumulus cells of ovine.2)The results of qRT-PCR and Western blot were basically consistent,the expression of Wnt2 mRNA and protein in granulosa cells with different sizes were significantly different(P<0.05),and the expression in large follicle granulosa cell was significantly higher than that in medium follicles(P<0.05),and the expression in medium follicle granulosa cell was significantly higher than that in small follicles(P<0.05).3)After silenced Wnt2 gene,the expressions of Wnt2 and CTNNB1 in Wnt2 siRNA group were significantly lower than those in nonsense sequence siRNA group(NC group)and blank control group(P<0.05),while the percentage of apoptosis in Wnt2 siRNA group was significantly higher than those in the other two groups(P<0.05).In conclusion,WNT2 can improve the biological function of ovaries granulosa cells in sheep by the WNT2/CTNNB1 signal pathway.