沉默Smad4基因表达抑制猪卵巢颗粒细胞增殖并使细胞G_1期阻滞

Smad4是TGF-β/Smad信号通路的核心下游信号分子.为探明Smad4基因对猪卵巢颗粒细胞增殖及细胞周期的影响,采用RNA干扰技术,设计并合成猪Smad4基因的靶向小分子干扰RNA,由LipofectamineTMRNAiMix介导转染体外培养的猪卵巢颗粒细胞.应用实时荧光定量PCR检测Smad4mRNA的干扰效果,应用MTT法、流式细胞术检测细胞增殖和细胞周期的变化,同时应用荧光定量PCR检测转染前后CyclinD1、CyclinB、CyclinA2、CDK1、CDK2、CDK4等周期相关基因的mRNA表达量的变化.实验结果显示,靶向猪Smad4的特异性siRNA序列对Smad4mRNA表达的抑制率为79.85%(P0.01);沉默Smad4可以显著抑制猪卵巢颗粒细胞增殖,并且改变细胞周期分布,G0/G1期细胞比例显著高于各对照组(P0.05),S期细胞比例显著低于各对照组(P0.05),细胞分裂被阻滞;转染36h后CyclinD1、CDK1的mRNA表达量显著低于对照组,CyclinA2、CDK2、CDK4极显著低于对照组,CyclinB差异不显著.综上所述,Smad4是影响猪卵巢颗粒细胞增殖及细胞周期进程的重要基因之一.

Silencing of Smad4 Induces G1-Arrest of the Cell Cycleto Inhibit the Proliferation of Porcine Granulosa Cells

Smad4 is an important molecule downstream of the TGF-β/Smad signaling pathway. To investigate the effect of Smad4-silencing on the proliferation and cell cycle in porcine granulosa cells, a Smad4-targeting small interference RNA( siRNA) was transfected into the cells using LipofectamineTM RNAi Mix. The Smad4 mRNA expression and the level of Cyclin D1, Cyclin B, Cyclin A2, CDK1, CDK2, CDK4 mRNA were assayed by real-time PCR at different stages of the cell cycle. MTT assays and flow cytometry were performed to analyze the cell proliferation and cell cycle. The results showed that a inhibition of Smad4 at 79.85% (P＜0.01) significantly altered the proliferation and cell cycle of the granulosa cells. The G0/G1 phase cells were increased, while S phase cells were decreased (P＜0.05) at 36 hours after transfection. The mRNA levels of Cyclin D1, CDK1 were decreased significantly (P＜0.05), as well as those of Cyclin A2, CDK2, CDK4 (P＜0.01),but the Cyclin B expression appeared to be stable.