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目的探讨生长分化因子-9(GDF9)对小鼠卵巢颗粒细胞增殖能力基因周期蛋白A(cyclin A)、周期蛋白D1(cyclin D1)和周期蛋白依赖性激酶抑制因子27(p27kip1)表达的影响。方法利用CCK-8检测法检测细胞的增殖能力;使用流式细胞术、Real-time PCR和Western blot法验证GDF9 siRNA的干扰效率;采用Real-time PCR和Western blot法检测细胞增殖cyclin A、cyclin D1和p27kip1 mRNA和蛋白表达情况。结果 GDF9 siRNA显著抑制颗粒细胞增殖活力,其中72 h抑制最明显(P<0.05);Real-time PCR和Western blot法检测结果显示GDF9 siRNA显著下调小鼠卵巢颗粒细胞中GDF9 mRNA及蛋白的表达(P<0.01);Realtime PCR法检测结果显示,细胞cyclin A、cyclin D1 mRNA表达降低,p27kip1 mRNA表达增强;Western blot法结果显示,GDF9 siRNA可促进细胞周期蛋白依赖性激酶抑制剂p27kip1的表达,同时下调G2/M期调控蛋白cyclin A、cyclin D1的表达。结论靶向抑制小鼠卵巢颗粒细胞中GDF9的表达,下调细胞cyclin A、cyclin D1的表达,上调P27 kip1的表达,显著抑制细胞的增殖。
Objective To investigate the effect of GDF9 on the expression of cyclin A, cyclin D1 and p27kip1 in mouse ovarian granulosa cells. Methods The proliferation of cells was detected by CCK-8 assay. The interference efficiency of GDF9 siRNA was verified by flow cytometry, Real-time PCR and Western blot. Real-time PCR and Western blot were used to detect the expression of cyclin A and cyclin D1 and p27kip1 mRNA and protein expression. Results GDF9 siRNA significantly inhibited the proliferation of granulosa cells (P <0.05). The results of Real-time PCR and Western blot showed that GDF9 siRNA significantly down-regulated the expression of GDF9 mRNA and protein in mouse ovarian granulosa cells ( P <0.01). The result of Realtime PCR showed that the expression of cyclin A and cyclin D1 mRNA was decreased and the expression of p27kip1 mRNA was enhanced. Western blot showed that GDF9 siRNA promoted the expression of cyclin-dependent kinase inhibitor p27kip1 Downregulate the expression of cyclin A and cyclin D1 in G2 / M phase. Conclusions Targeting inhibits the expression of GDF9 in mouse ovarian granulosa cells, down-regulates the expression of cyclin A and cyclin D1, up-regulates the expression of P27 kip1, and significantly inhibits the proliferation of mouse ovarian granulosa cells.