从“自噬-凋亡交叉对话”探讨补肾填精法对化疗性卵巢早衰卵泡内稳态的影响及分子机制

Premature ovarian failure(POF) induced by chemotherapy severely impair female quality of life. From the research of gap Junction and apoptotic pathway, we have already discovered that common prescription for tonifying kidney and replenishing essence Zuogui pill can effectively treat chemotherapy-induced POF. Autophagy is a concern for the maintenance of intracellular homeostasis in an abnormal environment. Newly published research reveals that autophagy exists in the follicle cells, and there was an interaction between the autophagy and apoptosis in the regulation of follicular development and atresia. The research will take ‘Cross-talking between autophagy and apoptosis’ as an entry point. Replicate the POF mouse model by cyclophosphamide injection. Transmission electron microscopy, laser scanning confocal microscope, Western-blot and RT-qPCR were used to analyze the expression of autophagy flow and its relationship with apoptosis, and to analyze the effect of chemotherapy on the homeostasis of follicles. To build the damage model of granulosa cells, oocytes and theca cells induced by Phosphoramide in vitro. Then silence autophagy genes by RNAi and enhance autophagy by transfecting siRNA. Analyze apoptosis genes by Western-blot and RT-PCR.. Analyze the cell cycle with how the cell cycle works in autophagy and apoptosis by flow cytometry. The research will expound above-mentioned interventional mechanism how Zuogui pill and drug serum interferes, so as to supply a new scientific theory for nourishing kidney-essence treating POF.

化疗诱发的卵巢早衰（POF）严重危害妇女生活质量。前期从细胞间缝隙通道与凋亡途径研究发现补肾填精法常用方剂左归丸能有效治疗化疗性POF。自噬作为细胞在异常环境中维持内稳态的生存方式备受关注。新近研究显示，卵泡内细胞存在自噬现象，与凋亡交互作用调节卵泡发育和闭锁。本项目拟“自噬-凋亡交叉对话”为切入点，通过环磷酰胺注射复制POF小鼠模型，透射电镜、激光扫描共聚焦显微镜、 Western-blot、RT-qPCR分析卵泡内细胞自噬流及其与凋亡间对话模式，分析化疗对卵泡内稳态的影响。通过磷酰胺氮芥体外诱导卵泡颗粒细胞、卵母细胞及卵泡膜细胞损伤模型，用RNA干扰沉默自噬基因、转染小干扰RNA质粒增强自噬，用 Western-blot、RT-qPCR分析凋亡基因；用流式分析细胞周期及其在自噬-凋亡中的关系。阐明左归丸及含药血清对上述的干预机制，为中医补肾填精法治疗POF补充新的科学理论。

化疗诱发的卵巢早衰（POF）严重危害妇女生活质量。自噬作为细胞在异常环境中维持内稳态的生存方式备受关注。自噬与凋亡交互作用调节卵泡发育和闭锁。本研究建立化疗性POF动物模型和体外细胞化疗性损伤模型，借助于透射电镜、激光扫描共聚焦显微镜、 Western-blot、qRT-PCR等进行检测自噬表达及其与细胞凋亡的关系。化疗性POF小鼠卵巢结构和功能受损，卵巢组织p62基因呈低表达，LC3B、Beclin-1、Caspase3和Bax基因呈高表达，经左归丸干预后，卵巢结构和功能好转，p62表达增加，LC3B、Beclin-1、Caspase3和Bax表达减少，并呈现量效关系，表明左归丸通过影响自噬和凋亡过程调节卵巢卵泡内稳态，恢复卵巢结构和功能。体外细胞实验进行了卵泡组分细胞的单培养和共培养。化疗损伤性颗粒细胞/膜细胞中，p62呈低表达，LC3B、Beclin-1、Caspase3和Bax呈高表达。左归丸含药血清可下调Beclin-1、LC3B、Bax和Caspase3模型组颗粒细胞/膜细胞中的表达，可上调模型组p62基因的表达，表明左归丸含药血清能缓解磷酰胺氮芥对颗粒细胞/膜细胞造成的损伤，同时影响了颗粒细胞/膜细胞自噬和凋亡过程。颗粒细胞实验同时表明在磷酰胺氮芥损伤颗粒细胞的过程和左归丸含药血清缓解其损伤的过程中均存在自噬与凋亡交叉对话。膜细胞也能缓解磷酰胺氮芥作用于颗粒细胞引起的化疗性损伤，且与左归丸含药血清具有协同作用。本研究为左归丸治疗化疗源性POF提供了实验依据。