沉默Gpr3基因表达对猪卵泡颗粒细胞凋亡的影响

Effects of Silencing Gpr3 Gene by Small Interfering RNA on Apoptosis in Porcine Granulosa Cells

G蛋白偶联受体3(G protein-coupled receptor 3,Gpr3)属于G蛋白偶联受体超家族成员,能够维持卵泡卵母细胞减数分裂的前期阻滞,但在卵泡颗粒细胞中的作用不清。该研究利用RNAi技术,以化学合成的siRNA转染体外培养的猪卵泡颗粒细胞,并利用Real-time PCR和Western blot技术检验Gpr3基因的沉默效果;利用MTT(四甲基偶氮唑盐)、流式细胞术和Real-time PCR技术检测沉默Gpr3基因表达对猪卵泡颗粒细胞凋亡以及凋亡相关基因表达的影响。结果显示,Gpr3-siRNA能够有效地抑制猪卵泡颗粒细胞中Gpr3基因mRNA和蛋白的表达(P<0.01);在沉默Gpr3基因表达后,猪卵泡颗粒细胞的细胞活性由0.419升高至0.586,同时细胞凋亡率由2.67%下降至0.42%,并在显著上调Bcl-2表达的同时,下调了Bax的表达(P<0.05)。结果表明,沉默Gpr3基因的表达抑制了猪卵泡颗粒细胞的凋亡,其机制可能与调控Bcl-2和Bax表达有关。

G protein-coupled receptor 3 （Gpr3）, a member of the G protein-coupled receptor superfamily, is known as a critical factor for the maintenance of meiotic prophase arrest in oocytes via a Gs protein-mediated pathway, but its role in granulosa cells is still unclear. In this study, RNAi was used to explore the effect of silenc- ing Gpr3 gene expression on apoptosis of porcine granulosa cells （GCs）. The specific siRNAs against Gpr3 was de- signed, chemically synthesized and transiently transfected into the procine GCs using lipidosome. Gpr3 expression level was assayed by Real-time PCR and Western blot. Cytoactive, apoptotic and the expression level of apoptotic- related genes were analyzed by MTT, flow cytometry and Real-time PCR, respectively. The results showed that Gpr3-siRNA effectively inhibited the expression of Gpr3 at both mRNA and protein levels in porcine GCs （P〈0.01）; the cytoactivity of porcine GCs increased from 0.419 to 0.586, and the apoptosis rate dropped from 2.67% to 0.42%; the expression levels of Bcl-2 were significantly increased, while the expression levels of Bax were reduced （P〈0.05）. Our results suggested that siRNA-mediated Gpr3 depletion inhibited apoptosis in porcine granulosa cells, which were possibly related to the expression levels of Bcl-2 and Bax genes.