SET在PCOS卵母细胞发育障碍中的功能研究

SET was one of the differential genes between PCOS ovaries and normal ones by cDNA microarray. Our previous studies confirmed that SET protein was higher in ovaries of PCOS patients compared to normal ovaries, and was predominantly localized in oocytes of PCOS ovaries and normal ovaries. It is found that SET participated in apoptosis regulation. Besids, SET overexpression cauesed precocious chromatid segregation, resulting in oocyte meiotic dysfunction. So it is hypothesized that SET may participate in oocyte developmental abnormality in PCOS. This study will investigate the oocyte maturation and apoptosis when SET was blocked by antibody or overexpressed and knockdown by recombinant adenoviruses. By testing the factors associated with oocyte maturation and apoptosis, the mechanism of SET on oocyte development will be elucidated. To elucidate whether SET participate in oocyte developmental abnormality in PCOS. This study will also study the maturation and apoptosis of immature oocyte from PCOS patiens when SET was blocked by antibody or knockdown by recombinant adenoviruses. By PP2A knockdown using recombinant adenoviruse and PP2A inhibitor and activators, the study will study the parthway of SET to regulates oocyte development. This study firstly investigate the effect of SET on oocyte development and the mechanism of SET in the pathogenesis of oocyte developmental abnormality in PCOS.

SET是通过基因芯片技术在多囊卵巢综合征和正常妇女卵巢中筛选出来的代表性差异基因，前期实验证实SET在PCOS卵巢中高表达且主要表达于卵母细胞。相关文献发现SET参与凋亡调节，且在卵母细胞中SET超表达可致减数分裂异常，推测SET可能参与PCOS卵母细胞发育障碍的病理生理过程。本研究利用卵母细胞体外培养模型，观察SET抗体阻断、表达抑制及超表达时小鼠卵母细胞成熟及凋亡情况，并检测成熟及凋亡相关因子mRNA、蛋白水平及酶活性的改变，阐明SET参与卵母细胞发育成熟的调节。观察SET抗体阻断及表达抑制时PCOS未成熟卵母细胞成熟及凋亡情况，验证SET参与PCOS卵母细胞发育障碍。通过PP2A干涉腺病毒，以及PP2A抑制剂及激动剂, 研究SET通过抑制PP2A调节卵母细胞发育成熟的信号机制。该课题首次研究SET在卵母细胞发育成熟中的调控机制，以及参与PCOS卵母细胞发育障碍的病理机制。

多囊卵巢综合征是育龄期妇女常见的内分泌及代谢紊乱性疾病，以高雄激素血症，卵巢多囊性改变和稀发排卵或无排卵为主要临床特征。卵泡发育异常是PCOS主要的病理特征，而PCOS患者卵巢中常存在卵母细胞发育障碍，提示PCOS患者卵泡发育异常与卵母细胞发育障碍有关。SET是通过基因芯片技术在多囊卵巢综合征和正常妇女卵巢中筛选出来的代表性差异基因，前期实验证实SET在PCOS卵巢中高表达且主要表达于卵母细胞。相关文献发现SET参与凋亡调节，且在卵母细胞中SET超表达可致减数分裂异常，推测PCOS患者中SET高表达可能导致卵母细胞发育障碍进而引起卵泡发育异常和排卵障碍，本研究探索了SET在PCOS卵母细胞发育障碍中的病理生理机制。. 本研究利用卵母细胞体外培养模型，观察SET表达抑制及超表达时小鼠卵母细胞成熟及凋亡情况，并检测成熟及凋亡相关因子的改变，阐明SET参与卵母细胞发育成熟 的调节。通过PP2A干涉腺病毒，以及PP2A抑制剂, 研究SET通过抑制PP2A调节卵母细胞发育成熟的信号机制。. 结果显示SET在GV期、MI期、MII期小鼠卵母细胞均有表达。干涉SET蛋白后卵母细胞的GVBD率及MII率均下降，卵母细胞成熟相关因子BMP 15、GDF 9亦下降；相反超表达SET后卵母细胞的GVBD率及MII率均上升，BMP 15、GDF 9亦上升。干涉SET后卵母细胞的凋亡率上升，卵母细胞凋亡相关因子Caspase 3、Caspase 9亦上升，Caspase 8及Cyt c无明显变化；相反超表达SET病毒后卵母细胞的凋亡率下降，卵母细胞凋亡相关因子Caspase 3、Caspase 9下降。我们进一步发现卵母细胞PP2A酶活性在超标达SET后被抑制并在干涉SET后增强。 PP2A的抑制剂冈田酸可致卵母细胞的GVBD率及MII率上升，干涉PP2A后卵母细胞的GVBD率及MII率亦上升。冈田酸处理后的卵母细胞可拮抗AdH1-SiRNA/SET介导的卵母细胞成熟抑制，进一步说明SET通过PP2A参与卵母细胞成熟调节。. 综上所述本研究揭示了SET参与小鼠卵母细胞的成熟及凋亡，并通过PP2A介导的特异性信号通路致小鼠卵母细胞成熟相关因子上升。结合我们先前的发现，SET在多囊卵巢综合征患者卵巢中高表达，推测SET参与多囊卵巢综合征的卵母细胞发育障碍。