Sirt1-Nrf2通路调节卵母细胞老化的机制研究

Oocyte quality decline plays a vital role in reducing elderly female reproductive ability. NF-E2 related factor 2 (Nrf2) is an age related transcription factor. Our previous studies have found that Nrf2 expression was decreased gradually with age in mouse oocyte and human granulosa cells; Sirt1-KD reduced expression of Nrf2, down-regulation of Nrf2 can significantly reduce the oocyte maturation rate, suggesting that Sirt1 may affect the oocyte meiosis and oocyte aging by regulation Nrf2. In vitro, to investigate the relationship between Sirt1 and Nrf2 ,and its downstream pathway in oocyte aging, the expression of Sirt1 and Nrf2, the effects of Sirt1 and Nrf2 on oocyte maturation, spindle morphology, chromosome aneuploidy and cyclin expression were studied in mouse oocytes, which can elucidate the role of Sirt1-Nrf2 pathway in oocyte meiosis and aging. Then the effect of Nrf2 on the aging oocytes was verified by experiments in vivo. The present results will help us to improve oocyte quality of aged women to provide new theoretical basis and ideas.

卵母细胞质量下降是导致高龄女性生殖能力减退的重要原因。Nrf2是一种与年龄密切相关的核转录因子，本课题组前期研究发现，Nrf2在人颗粒细胞和小鼠卵子中的表达水平与年龄呈显著负相关，小鼠卵母细胞中Sirt1-KD能降低Nrf2的表达，下调Nrf2能够明显降低卵子成熟率，提示Sirt1可能通过Nrf2影响卵母细胞减数分裂并参与卵子老化过程。为此本项目拟从临床层面，以人颗粒细胞为模型，研究Sirt1和Nrf2及其下游通路与年龄的相关性；在体外细胞层面，通过调节小鼠卵母细胞中Sirt1和Nrf2的表达，探讨Sirt1与Nrf2的关系及其对卵母细胞成熟、纺锤体形态、染色体非整倍性、细胞周期蛋白表达等的影响，阐明Sirt1-Nrf2通路在卵子减数分裂及老化中的作用和分子机制；通过动物体内实验，验证Nrf2对卵母细胞老化的调节作用，为提高高龄妇女卵母细胞质量提供新的思路和理论依据。

卵母细胞是构成雌性动物生殖能力的核心，高质量卵母细胞对胚胎的正常发育和健康后代的产生十分重要。卵母细胞质量下降是导致高龄女性生殖能力减退的重要原因。Nrf2是一种重要的一种与年龄密切相关转录因子，广泛参与细胞内各种生理活动，但其在减数分裂过程中的作用鲜有报道。我们首先通过使用Nrf2的抑制剂Brusatol初步研究Nrf2在卵母细胞减数分裂中的可能的作用通路。发现Brusatol处理能够通过对Nrf2水平的调节影响Cyclin B1的表达，并可能通过这一通路调控卵母细胞减数分裂进程。随后通过显微注射siRNA的方法调节小鼠卵母细胞中Sirt1和Nrf2的表达，探讨Sirt1与Nrf2的关系及其对卵母细胞成熟、纺锤体形态、染色体非整倍性、细胞周期蛋白表达等的影响。结果发现Sirt1敲低能降低Nrf2表达，证明小鼠卵母细胞中存在Sirt1-Nrf2-Cyclin B1信号通路。另外，我们发现来自老年小鼠的卵母细胞的Nrf2蛋白水平较低，并且通过Nrf2的过表达可以改善母体年龄相关的减数分裂缺陷，提示了Nrf2的表达减少是导致卵母细胞老化的因素之一。总之，我们的数据表明Nrf2在卵母细胞减数分裂中具有重要的作用，并揭示了增加的Nrf2表达对老年卵母细胞的显着的有益效果，为提高高龄妇女卵母细胞质量提供新的思路和理论依据。