抑制BMP/Smad信号通路对水牛卵巢颗粒细胞生长和类固醇激素合成的影响

Effects of Inhibition of BMP/Smad Signaling Pathway on the Growth and Steroid Hormone Synthesis of Buffalo Ovarian Granulosa Cells

【目的】探究BMP/Smad信号通路对水牛卵巢颗粒细胞生长和类固醇激素合成的影响。【方法】利用脂质体转染的方法将合成的Smad 4基因的3对siRNAs(Smad4-siRNA1、Smad4-siRNA2和Smad4-siRNA3)和NC-siRNA(对照组)分别转染水牛颗粒细胞,通过实时荧光定量PCR检测Smad 4基因的表达水平,比较不同siRNA的干扰效率。然后利用筛选的干扰效率最高的siRNA,转染水牛卵巢颗粒细胞,通过CCK-8法检测对照组和干扰组细胞的增殖情况,实时荧光定量PCR检测上述两组细胞凋亡相关基因Bax和Bcl2,细胞周期相关调控基因CyclinD2和CDK4,以及类固醇激素合成相关基因Cyp19A1和Cyp11A1的表达水平,并通过ELISA检测雌二醇(E 2)和孕酮(P 4)的含量。【结果】基因干扰试验结果表明,3对siRNAs对Smad4基因的表达均具有极显著的干扰作用(P<0.01),其中Smad4-siRNA1的干扰效率最高,达到了64%。CCK-8检测结果显示,与对照组相比,干扰组细胞的增殖效率显著降低(P<0.05)。实时荧光定量PCR结果显示,与对照组相比,干扰组Bcl2、CyclinD2、CDK4和Cyp19A1基因的表达量均显著或极显著下调(P<0.05;P<0.01),而Cyp11A1基因的表达极显著上调(P<0.01),Bax基因的表达未受影响。ELISA检测结果表明,与对照组相比,干扰组雌二醇分泌量减少10.2%,孕酮的分泌量增加24.7%,差异均显著(P<0.05)。【结论】通过RNAi介导的Smad 4基因沉默对内源性BMP/Smad信号通路的中断不仅能够显著抑制水牛颗粒细胞的生长,而且能够诱导水牛颗粒细胞的凋亡,还会改变类固醇激素生成。BMP/Smad信号通路在调控水牛颗粒细胞生长和类固醇生成过程中具有重要的作用。

【Objective】The aim of this study was to investigate the effect of BMP/Smad signaling pathway on the growth and steroid hormone synthesis of buffalo ovarian granulosa cells.【Method】Three pairs siRNAs of Smad 4 gene(Smad4-siRNA1,Smad4-siRNA2 and Smad4-siRNA3)and NC-siRNA(control group)were transfected into buffalo granulosa cells by lipofection.The expression level of Smad 4 gene was detected by Real-time quantitative PCR to compare the interference efficiency of different siRNAs.Then the siRNA with the highest interference efficiency was transfected into buffalo ovarian granulosa cells.The cell proliferation in the control group and interference group was detected by CCK-8,and the expression of the apoptosis-related genes Bax and Bcl2,cell cycle-related regulation genes CyclinD2 and CDK4 and the steroid hormone synthesis related genes Cyp19A1 and Cyp11A1 were also detected by Real-time quantitative PCR,and the contents of estradiol(E 2)and progesterone(P 4)were detected by ELISA.【Result】The results of gene interference test showed that the three pairs of siRNAs had extremely significant interference effects on the expression of Smad 4 gene(P<0.01),and the interference efficiency of Smad4-siRNA1 was the highest,reaching 64%.The CCK-8 test results showed that compared with control group,the cell proliferation rate of the interference group was significantly decreased(P<0.05).The results of Real-time quantitative PCR showed that compared with the control group,the expression of Bcl2,CyclinD2,CDK4 and Cyp19A1 genes in the interference group were significantly or extremely significantly down-regulated(P<0.05 or P<0.01),while the expression of Cyp11A1 gene was extremely significantly up-regulated(P<0.01),and the expression of Bax gene was not affected.The results of ELISA showed that compared with the control group,the estradiol secretion in the interference group decreased by 10.2%,and the progesterone secretion increased by 24.7%,and the difference was significant(P<0.05).【Conclusion】The disruption of endogenous BMP/Smad signaling pathway by RNAi-mediated Smad 4 gene silencing could not only significantly inhibit the growth of buffalo granulosa cells,but also induce the apoptosis of buffalo granulosa cells.Furthermore,inhibiting BMP/Smad signal would change the production of steroid hormones.BMP/Smad signal pathway played an important role in regulating the growth of buffalo granulosa cells and the production of steroids.