沉默Smad8基因对小鼠卵泡颗粒细胞增殖的影响

Effect of Silence Smad8 Gene on Proliferation of Granulosa Cells in Mice

SMAD8(又称SMAD9)蛋白质是TGF-β/SMADs信号通路中重要的转录因子。该研究利用RNA干扰技术沉默Smad8基因,探讨该基因对小鼠卵泡颗粒细胞增殖的影响。免疫组化技术对SMAD8在小鼠卵泡的表达进行定位,设计并合成Smad8-si RNA转染小鼠颗粒细胞,荧光定量PCR(q PCR)和Western blot检测Smad8基因沉默效率,CCK-8法分析颗粒细胞增殖能力,ELISA检测细胞上清中雌二醇(E2)和孕酮(P4)浓度,q PCR检测颗粒细胞促卵泡素受体(follicle stimulating hormone receptor,FSHR)、促黄体素受体(luteinizing hormone receptor,LHR)以及与细胞增殖相关的细胞周期调控蛋白基因Cyclin D2和CDK4 m RNA水平。结果显示,SMAD8仅表达于卵泡的颗粒细胞,Smad8-si RNA有效抑制了Smad8的表达(P<0.01),Smad8沉默后颗粒细胞的增殖能力明显减弱,细胞上清中E2水平显著下降,P4水平未受影响,颗粒细胞LHR、Cyclin D2和CDK4 m RNA水平明显降低,FSHR m RNA无明显变化。以上结果表明,沉默Smad8基因降低了小鼠颗粒细胞的增殖能力,其机制可能与沉默Smad8调低了颗粒细胞增殖分化相关的E2合成以及LHR、Cyclin D2和CDK4的表达下降有关。

SMAD8（also named SMAD9） protein is one of the important transcription factorsin TGF-β/SMADs signaling pathway. In this study, RNAi was used to explore the effect of silencing Smad8 gene expression on proliferation of mouse granulosa cells（MGCs）. Immunohistochemistry was used to locate SMAD8 expression in mouse follicle. Smad8 gene si RNA was designed and synthesized to transfect MGCs and Smad8 gene was detected by q PCR and Western blot after transfection with Smad8-si RNA. Cell proliferation capacity was analyzed by CCK-8 and the concentrations of E2 and P4 in supernatant were determined by ELISA. The q PCR was performed to measure FSHR, LHR, Cyclin D2, CDK4 m RNA levels in granulosa cells after transfection with Smad8-si RNA. The results showed that Smad8 gene expression was effectively inhibited by Smad8-si RNA（P〈0.01）, cell proliferation capacity was significantly weakened. E2, LHR, Cyclin D2 and CDK4 m RNA levels were significantly decreased, but P4 and FSHR m RNA remain unchanged. These results indicated that the silencing Smad8 gene decreased the proliferation ability of MGCs, and its mechanism might be related to the decline of E2 secretion and LHR, Cyclin D2, CDK4 expression decrease after Smad8 gene silence.