卵巢低反应女性中沉默信息调节因子1对卵巢颗粒细胞的调控

Regulation of ovarian granulosa cell by SIRT1 in women with poor ovarian response

目的探讨卵巢低反应女性中沉默信息调节因子1(SIRT1)对卵巢颗粒细胞的调控。方法本研究纳入行IVF/ICSI-ET助孕的女性共60例,其中卵巢低反应(POR)组和卵巢正常反应(NOR)组各30例。采用密度梯度离心法分离纯化卵泡液中的颗粒细胞。分别使用SIRT1激动剂白藜芦醇(RESV)或抑制剂EX527处理POR组颗粒细胞,对照组为不使用药物处理的颗粒细胞。采用qPCR检测颗粒细胞SIRT1和类固醇激素合成关键酶(StAR、CYP19、CYP17)的mRNA水平。采用Western blot检测颗粒细胞SIRT1、类固醇激素合成关键酶蛋白和凋亡相关蛋白(Bcl-2、Caspase-3)的水平。TUNEL法检测颗粒细胞的凋亡率。化学发光法检测颗粒细胞培养液中雌二醇水平。结果POR组颗粒细胞SIRT1 mRNA和蛋白水平低于NOR组(P=0.003,P<0.001)。RESV上调SIRT1 mRNA和蛋白水平(P<0.001),上调类固醇激素合成关键酶mRNA和蛋白水平(P<0.05),EX527下调SIRT1 mRNA和蛋白水平(P=0.023、0.001),下调类固醇激素合成关键酶mRNA和蛋白水平(P<0.05)。RESV作用颗粒细胞后,细胞凋亡率降低(P=0.000),Bcl-2蛋白水平上升(P<0.001),Caspase-3蛋白水平下降(P<0.001);EX527作用颗粒细胞后,细胞凋亡率升高(P<0.001),Bcl-2蛋白水平下降(P=0.003),Caspase-3蛋白水平上升(P<0.001)。结论POR女性颗粒细胞SIRT1水平低于NOR女性。RESV增加SIRT1表达。SIRT1抑制颗粒细胞凋亡,提高类固醇激素合成关键酶水平,促进颗粒细胞合成雌二醇。因此,SIRT1激活剂如RESV可能成为改善POR女性卵巢反应性,增加获卵数的治疗药物。

Objective To investigate the regulation of SIRT1 on ovarian granulosa cell in women with poor ovarian response.Methods A total of 60 women who underwent IVF/ICSI-ET were included in this study,30 women in poor ovarian response(POR)group and 30 women in normal ovarian response(NOR)group.The granulosa cells in follicular fluid were isolated and purified by density gradient centrifugation.The granulosa cells of POR group were treated with SIRT1 agonist resveratrol(RESV)or inhibitor EX527,respectively,and the control group was granulosa cells treated without drugs.mRNA levels of SIRT1 and key enzymes of steroid hormone synthesis(StAR,CYP19,CYP17)in granulosa cells were detected by qPCR.The levels of SIRT1,steroid hormone synthesis key enzymes protein and apoptosis-related protein(Bcl-2,Caspase-3)in granulosa cells were detected by Western blot.The apoptosis rate of granulosa cells was detected by TUNEL method.Determination of estradiol in granulosa cell culture medium by chemiluminescence.Results The mRNA and protein levels of SIRT1 in granulosa cells in POR group were lower than those in NOR group(P=0.003,P<0.001).RESV up-regulated SIRT1 mRNA and protein levels(P<0.001),up-regulated mRNA and protein levels of steroid hormone synthesis key enzyme(P<0.05).EX527 down-regulated SIRT1 mRNA and protein levels(P=0.023,0.001),down-regulated mRNA and protein levels of steroid hormone synthesis key enzymes(P<0.05).After RESV treatment,the apoptosis rate of granulosa cells was decreased(P<0.001),the Bcl-2 protein level was increased(P<0.001),and the Caspase-3 protein level was decreased(P<0.001).After EX527 treatment,the apoptosis rate of granulosa cells was increased(P<0.001),the Bcl-2 protein level was decreased(P=0.003),and the Caspase-3 protein level was increased(P<0.001).Conclusion The SIRT1 level in granulosa cells of POR women was lower than that of NOR women.RESV up-regulated SIRT1 expression in granulosa cells.SIRT1 inhibited granulosa cell apoptosis,increased the level of steroid hormone synthesis key enzyme,and promoted estradiol synthesis in granulosa cell.Therefore,SIRT1 activators such as RESV may be a therapeutic agent to improve ovarian reactivity and increase the number of oocytes obtained in women with POR.