Zhangfei介导的内质网应激在卵泡闭锁中的作用及其调控机制

In mammalian ovaries, more than 99% of follicles undergo a degenerative process known as atresia. Apoptosis of ovarian granulosa cells plays a critical role in follicular atresia.Thus,it is a major target to reveal the regulation mechanism of granulosa cell apoptosis in follicular atresia study.Recent .studies have identified a new apoptosis signaling pathway, endoplasmic reticulum stress (ERS)-induced apoptosis. However, the potential role of this pathway in regulating follicular development and atresia has not been reported. Zhangfei is an important transcription factor in the ERS pathway, and previous studies in our laboratory revealed a possible relationship between Zhangfei and granulosa cell apoptosis,but the detailed roles and mechanisms remain largely unknown. In this study, immunohistochemistry, in situ hybridization, TUNEL, real-time PCR, western blotting, overexpression and RNAi technique were used to investigate whether ERS-induced apoptosis is involved in granulosa cell apoptosis during follicular atresia. We also established an in vitro ERS model of mouse granulosa cells to confirm the exact roles and molecular mechanism of Zhangfei during granulosa cell apoptosis. Our results would reveal a new pathway responsible for the regulation of granulosa cell apoptosis, and provide a new research direction for further investigation of the mechanisms regulating follicular development and atresia.

哺乳动物卵巢上99％以上卵泡归于闭锁，颗粒细胞凋亡是导致卵泡闭锁的关键，揭示颗粒细胞凋亡的调控机制是研究卵泡闭锁的核心。内质网应激（ERS）诱导的细胞凋亡是一类新的凋亡通路，迄今尚未见该通路调控卵泡发育与闭锁的相关报道。Zhangfei是ERS通路中的重要转录因子，我们发现Zhangfei与颗粒细胞凋亡的发生相关，但其具体作用及其分子机制有待阐明。本项目拟采用免疫组化、原位杂交、TUNEL、Real-time PCR、Western Blot及基因过表达和RNAi技术等，在明确ERS相关的凋亡通路参与调节小鼠卵泡闭锁进程的基础上，进一步构建小鼠颗粒细胞ERS模型，阐明Zhangfei在颗粒细胞凋亡发生、发展中的作用及由ERS介导的相关信号转导通路，揭示颗粒细胞凋亡的新机制，为全面认识卵泡发育与闭锁的调控机理提供新思路和实验依据。

哺乳动物卵巢上99％以上卵泡归于闭锁，颗粒细胞凋亡是导致卵泡闭锁的关键，揭示颗粒细胞凋亡的调控机制是研究卵泡闭锁的核心。内质网应激（ERS）诱导的细胞凋亡是一类新的凋亡通路，本实验室前期研究证实，ERS通路参与山羊卵泡发育与闭锁的调控过程。CREBZF是ERS通路中的重要转录因子，预实验中我们发现CREBZF与小鼠颗粒细胞凋亡的发生相关，因此，本项目以期在明确ERS相关的凋亡通路参与调节小鼠卵泡闭锁进程的基础上，通过构建小鼠颗粒细胞ERS模型，阐明CREBZF在颗粒细胞凋亡发生、发展中的作用及由ERS介导的相关信号转导通路。本项目成功构建了针对小鼠CREBZF基因的过表达和shRNA干扰重组慢病毒载体，为进一步研究CREBZF在小鼠机体中的生理功能奠定了基础；应用Real-time PCR、免疫组织化学和Western Blot技术揭示了CREBZF基因在小鼠卵泡发育和闭锁过程中的分布特点和表达规律；在完善小鼠卵泡颗粒细胞体外内质网应激（ERS）模型的基础上，应用过表达和shRNA干扰技术分析了CREBZF基因对小鼠颗粒细胞增殖和凋亡的影响；进一步检测分析了CREBZF基因通过未折叠蛋白信号通路调控小鼠卵泡闭锁的作用机制。本研究揭示了颗粒细胞凋亡的新机制，为全面认识卵泡发育与闭锁的调控机理提供新思路和实验依据。