人羊膜上皮细胞外泌体miR-2861对化疗诱导原始卵泡激活 Kit／Kit Ligand 通路的分子调控机制

Chemotherapeutic agents are used to treat a wide variety of malignancies, and unavoidably cause progressive ovarian damage that can cause reduced fertility in young female patients.Previous studies have demonstrated that transplantation of hAEC or hAECs derived conditioned medium (hAECs-CM) could effectively alleviate chemotherapy-induced ovarian damage by inhibiting granulosa cells (GCs) apoptosis and promoting follicle development in animal models of premature ovarian failure and insufficiency (POF/POI). The mechanisms of benefit are known to be indirect, but the mediators have yet to be identified.Exosomes have emerged as a new avenue for cell-to-cell communication and an important component of paracrine pathway. A more interesting consideration is that stem cells could secrete exosomes, which play an important role in microRNA communication between donor cells and recipient tissues. In the current study, we mainly investigate the biological function of hAECs-derived exosomes (hAECs-exo) and whether they as a molecular basis regulate follicle activation induced by chemotherapy. In addition, we will detect the effect of exosomal miRNA-2861 derived from hAECs on follicle activation and analyze target gene of miRNA2861 in the process of follicle activation. Finally,our study will further discuss that exosomal miRNA-2861 derived from hAECs may offer a viable method for treating chemotherapy-induced ovarian injury.These novel insights offer a clue to the potential mechanism underlying hAECs promoting ovary function repair, which may be able to preserve the fertility in female cancer patients.

化疗药物作为治疗肿瘤的常用方法，是导致女性肿瘤患者生育能力下降的主要原因。人羊膜上皮细胞移植具有修复化疗后卵巢功能的作用，主要表现为抑制化疗诱导的颗粒细胞凋亡及促进卵泡发育与成熟。前期研究发现，其旁分泌途径在其修复卵巢功能过程中发挥重要作用，但具体分子机制并不清楚。外泌体是一种细胞与细胞之间交流的重要介质，干细胞旁分泌途径中的重要组成部分。干细胞分泌的外泌体通过传递特定的microRNAs调控受体细胞的功能并参与组织损伤的修复过程。在本研究中，首先分析人羊膜上皮细胞来源外泌体的生物学功能以及其对化疗药物诱导的原始卵泡激活过程中Kit/Kit Ligand 信号通路的影响。此外，分析外泌体中miRNA-2861的表达及其对化疗诱导原始卵泡激活过程中靶基因Kitlg的分子调控机制。最后，探讨人羊膜上皮细胞是否可作为一种载体通过传递miRNA-2861进一步提高修复卵巢功能的能力。

卵巢早衰是女性肿瘤患者在接受化疗药物治疗后常见的并发症之一。越来越多的研究表明，人羊膜上皮细胞在多种疾病或组织器官损伤中具有良好的修复作用。前期研究发现，人羊膜上皮细胞在化疗诱导的卵巢早衰中具有修复卵巢功能的潜能，且主要是依赖于其旁分泌途径实现。本研究主要对人羊膜上皮细胞分泌的外泌体进行分离及鉴定，并研究人羊膜上皮细胞来源的外泌体在化疗药物诱导的卵巢损伤小鼠模型中的修复作用，及探讨可能的分子机制。研究发现，人羊膜上皮细胞来源的外泌体呈现典型的杯状结构，粒径约在100nm左右，且高表达外泌体特异性蛋白Alix，CD63和CD9。在卵巢早衰的小鼠模型中，人羊膜上皮细胞外泌体移植后显著增加损伤卵巢内的卵泡数量并改善卵巢功能。在移植早期，人羊膜上皮细胞外泌体具有抑制化疗药物诱导的颗粒细胞凋亡并减轻急性血管损伤的作用，同时抑制化疗药物诱导的原始卵泡过度激活。microRNAs表达谱分析显示，在人羊膜上皮细胞外泌体中存在大量具有生物学功能的microRNAs，经生物信息学分析发现大量的microRNA调控的靶基因主要富集在磷脂酰肌醇信号转导通路（PI3K）和凋亡调控途径。进一步的离体细胞实验证实人羊膜上皮细胞分泌的外泌体能够被卵巢颗粒细胞所摄取，通过传递功能性microRNAs（例如miR-1246及miR-21-5p）抑制化疗药物诱导的颗粒细胞凋亡的发生。本研究首次阐明了人羊膜上皮细胞来源的外泌体具有修复化疗后小鼠卵巢功能的作用，具体的分子机制是通过传递功能性的microRNAs实现。本研究不仅为阐明以人羊膜上皮细胞为基础的卵巢功能修复提供了重要的科学依据，同时也为卵巢早衰的去细胞化生物治疗提供了新的思路。