利用UFL1基因敲除小鼠探讨颗粒细胞内ER stress对卵泡发育的影响及机制研究

The E3 ligase UFL1, one of Ubiquitin-like modified system molecular, has been shown to induce multiple intracellular ER stress when it was deleted, which may affect cell proliferation or apoptosis. Ovarian GC proliferation, differentiation and apoptosis play a key role in follicular development. Our pre-experiments showed that Knockout of UFL1 expression leads to apoptosis of GC and increase of atresia follicles, suggesting that UFL1 deletion causes ER stress in GC and leads to follicular atresia. By Construct UFL1-/- knock down mouse with UFL1Loxp/Loxp mouse mating with AMH-Cre, Rosa-Cre-ERT2 under different conditions, our projects intend to explore the mechanism of ER stress in GC and the influence of follicle development absence URFL1. The UFL1-/- GC, follicular development and steroidal hormones will be detected by TUNEL, HE, Elisa; the primary culture UFL1-/- GC will be used to detect the markers of ER stress, proliferation, differentiation and apoptosis by qPCR, IB, and GC proliferation, differentiation and apoptosis will be detected by CCK8,FCM; Lentivirus UFL1, FSH and ER stress inhibitor are used to observe the rescue of ER stress in UFL1-/- GC; and Lentivirus UFL1-shRNA infection is used to induce ER stress in wild type GC.

UFM1类泛素化修饰系统成员E3连接酶UFL1的缺失被证实可引发多种细胞内ER stress，影响细胞增殖或凋亡。卵巢GC增殖、分化和凋亡对卵泡发育起关键作用。我们前期实验发现，敲除UFL1表达造成GC凋亡，闭锁卵泡增多，提示UFL1缺失引发了GC内ER stress，导致卵泡闭锁。本课题拟构建UFL1-/-转基因小鼠，探讨敲除UFL1后造成GC内ER stress，影响卵泡发育的机制。摘取UFL1-/-卵巢，TUNEL、HE，ELISA等检测卵泡内GC，卵泡发育及甾体激素；原代分离GC，qPCR、IB检测ER stress、增殖、分化和凋亡标志分子，CCK8、FCM等检测GC增殖、分化和凋亡；采用UFL1过表达慢病毒、FSH及ER stress阻断剂，观察对GC内ER stress的拯救；采用UFL1-shRNA慢病毒感染WT GC，观察引发ER stress现象。

卵巢内颗粒细胞（Granular cell，GC）的增殖和凋亡是卵泡启动和发育的决定性因素之一。Ufmylation类泛素化系统E3连接酶UFL1的表达缺失被证实可激发多种细胞的内网质应激（ER stress），进而影响细胞增殖或凋亡，但UFL1是否也通过调控GC的增殖或凋亡进而影响卵巢功能仍未明确。本项目中，拟分别在组织和细胞水平敲除GC内UFL1表达后，研究是否造成GC内ER stress，影响GC的增殖和凋亡，进而影响卵巢内各级卵泡的发育和数量；进一步研究UFL1在化疗药物及炎症刺激下是否对GC、卵泡发育和卵巢功能具有保护作用；最后探讨UFL1在卵巢早衰（Premature ovarian failure，POF）中的可能机制以及对雌性生殖功能的影响等。我们结果显示，UFL1的表达水平随小鼠生殖年龄增加而下降，在顺铂诱导的POF小鼠内表达降低，UFL1表达缺失加剧了顺铂诱导的卵巢GC的ER stress和细胞凋亡，而过表达UFL1能缓解顺铂诱导的ER stress和细胞凋亡。在体外培养卵巢内，当敲除UFL1的表达时会导致各级卵泡数量下降以及卵泡闭锁率上升，而过表达UFL1则可减轻顺铂诱导的POF，卵泡数量得以部分恢复，改善卵泡闭锁情况。另外，在人卵巢颗粒细胞瘤细胞系KGN内，UFL1可以缓解脂多糖（LPS）刺激引发的KGN内ER stress现象，当在KGN内敲除UFL1时会触发的铁死亡和氧化应激，提示UFL1对KGN的保护机制可能与ER stress和铁死亡相关。通过本课题的实施，我们阐明了在卵巢、GC和KGN内，UFL1的表达缺失可激发或加剧ER stress，导致卵泡发育障碍和数量下降，生殖功能受损，UFL1对化疗药物和炎症导致的POF具有保护作用，我们的研究为临床治疗不育不孕、恶性肿瘤化疗导致的生殖损伤及卵巢早衰提供新的思路、方法和药物靶点。