卵巢玻璃化冷冻移植中内质网应激诱导凋亡的分子机制研究

Autotransplantation of cryopreserved ovarian is one of the effective methods to preserve fertility of young cancers, however hypoxic ischemia after cryopreservation and transplantation can lead to apoptosis of most follicles. Endoplasmic reticulum stress plays a important role on tissue apoptosis of hypoxic ischemia after organs transplantation. In our previous study, cryopreservation led to increased apoptosis and elevated expression of GRP78 (endoplasmic reticulum chaperone ) and CHOP (apoptotic protease-activating factor), in vitro intervention with follicle stimulating hormone (FSH) could inhibit this phenomenon. So it is inferred that cryopreservation and post-transplant ischemia and hypoxia could lead to endoplasmic reticulum stress and then induce apoptosis. In this project，through approaches of real-time PCR, western blotting and immunohistochemistry and so on, we plan to clarify the role and mechanism of the endoplasmic reticulum stress (ERS) in the process of ovarian cryopreservation and transplantation by detecting the expression level and/or activity of ERS characteristic molecules(such as GRP78, IRE1, XBP1, PERK and eIF2, etc.) and its downstream apoptotic signaling pathway markers (such as caspase12, CHOP/GADD153, c-Jun,etc.) in ovarian tissue, and observing the effects that ERS acts to the transplanted ovary, and furthermore, to explore the effects of FSH on ERS and its molecular mechanism. Our study will lay foundation for clinical implication of ovarian cryopreservation and transplantation.

冻存卵巢的自体移植是保存青年癌症患者生育力的策略之一，但冻存损伤和移植后的缺血缺氧会导致卵泡大量凋亡。内质网应激（ERS）是移植器官缺血缺氧损伤时造成组织凋亡的主要途径。项目组前期工作表明，冷冻导致内质网伴侣蛋白GRP78和凋亡激活因子CHOP表达升高、凋亡增加，卵泡刺激素(FSH)体外干预可抑制其高表达，降低凋亡。由此推测卵巢冻存及移植后的缺血缺氧损伤可能激活ERS并诱导凋亡发生。本项目拟通过实时荧光定量PCR技术、免疫印迹技术和免疫组化等方法检测卵巢组织ERS的标志性分子GRP78,IRE1,XBP1,PERK和eIF2等及其下游凋亡信号通路的关键标志分子caspase12,CHOP/GADD153,c-Jun等的表达水平或活性状态，观察ERS对移植卵巢功能的影响，阐明ERS在卵巢冷冻移植中的作用及机制；并探索FSH干预对ERS的影响及分子机制，为卵巢冷冻移植的临床应用奠定实验室基础。

冻存卵巢的自体移植是保存青年癌症患者生育力的策略之一，但冻存损伤和移植后的缺血缺氧会导致卵泡大量凋亡。本研究以4周龄青春期雌性小鼠卵巢为模型，以项目组自创的玻璃化冷冻模式进行冷冻，移植卵巢行肾被膜下移植，以TM诱导作为内质网应激（ERS）阳性对照，TUDCA作为ERS抑制剂，探讨ERS在卵巢冷冻移植过程中的作用及其机制。结果显示，卵巢冷冻过程中的缺血缺氧损伤启动了ERS，内质网应激伴侣分子GRP78表达明显高于新鲜对照组（P<0.05），并由此引发UPR反应而启动了卵泡的凋亡，ERS相关的凋亡蛋白CHOP、caspase-12表达水平升高（P<0.05）。缺血缺氧可能主要是通过ERS途径诱导了原始卵泡的闭锁。卵巢冷冻移植的过程中，内质网应激主要发生在生长早期，在移植后的第2天最强; 发育的第4天，内质网应激保护途径已经转向了凋亡途径。移植后的卵泡发育过程中的卵泡丢失跟内质网应激相关。卵巢冷冻过程中FSH干预可通过调控ERS途径减少卵泡凋亡。FSH干预可明显改善卵泡形态和卵巢功能的恢复，缩短移植后动情周期恢复时间。受体腹腔注射ERS抑制剂在移植的早期对原始卵泡有一定的保护作用，可以减少原始卵泡的闭锁，并促进卵泡发育。研究结果为卵巢冷冻移植的临床应用奠定实验室基础。