脂肪间充质干细胞外泌体修复化疗致卵巢早衰大鼠作用及机制研究

Premature ovarian failure (POF) , which lead to infertility and perimenopausal syndrome symptoms and affect women's reproductive health and family and social stability seriously, is an important and common long-term adverse event of curative chemotherapy. Until recently, there had been limited progress in the field of recovery of ovulation and ovarian endocrine function fundamentally. Mesenchymal stem cells (MSCs) and conditioned medium can repair POF induced by chemotherapy, to restore endocrine function, treatment of infertility, MSCs play a major role mainly by paracrine. MSCs-derived exosomes are the membrane secretion system released by MSCs in the resting or activated state into the extracellular metrix. Exosomes contain not only active substances of MSCs, but also possess the advantages of selective assembly, targeting delivery, security , stability and large-scale production, witch shows a huge advantage in the repair of tissue regeneration. .The purpose of this project is to evaluate the feasibility of treatment of adipose mesenchymal stem cells exosomes on rats damage granulosa cells and POF due to chemotherapy. To investigate the basis of the therapeutic benefit of MSCs exosomes, we will compare their miRNA repertoire to that of fibroblasts exosomes using a PCR microarray. Whole-transcriptome microarrays is also processed of damage granulosa cells after exosomes treament. According to our study, we can get a new and safe method in POF repair.

卵巢早衰（POF）是肿瘤化学药物治疗严重后遗症，会导致不孕，围绝经期综合征症状，严重影响女性生殖健康和家庭及社会稳定。目前缺乏从根本上恢复卵巢内分泌及排卵功能方法。间充质干细胞（MSCs）及条件培养液可以修复化疗导致的POF，恢复内分泌功能，治疗不孕，MSCs主要通过旁分泌途径发挥作用。间充质干细胞外泌体是MSCs在静息或活化状态下释放到细胞外基质中的膜分泌体系，含MSCs的活性物质，具有选择性组装、靶向性投递、安全性高、化学性质稳定等优点，可规模化生产保存，在组织再生修复中显示了巨大的优势。.本项目通过体内体外实验研究脂肪间充质干细胞外泌体对化疗损伤颗粒细胞及化疗致POF大鼠的修复作用，利用测序技术分析修复后颗粒细胞转录组变化情况，并与脂肪间充质干细胞外泌体miRNA表达谱关联分析，对相关miRNA进行功能分析，探索外泌体修复POF的机制。为建立一种安全有效的POF治疗新方法奠定基础。

卵巢早衰（POF）是肿瘤放化疗严重后遗症，会导致卵巢功能减退，围绝经期综合征，严重影响女性生殖健康和家庭及社会稳定。前期研究发现间充质干细胞（MSCs）及条件培养液可以修复放化疗导致的大鼠卵巢早衰，恢复内分泌功能，MSCs主要通过旁分泌途径发挥作用。.本项目首先通过全身放疗制备了POF大鼠，放疗组大鼠放疗后血清中性激素E2和AMH表达水平较对照组明显降低；卵巢组织HE染色后观察并进行卵泡计数，放疗组各级卵泡特别是生长阶段卵泡数量明显减少，成功制备放疗致POF大鼠模型。进一步对脂肪间充质干细胞(A-MSCs)条件培养液中的外泌体进行分离鉴定，然后将A-MSCs外泌体对放疗致POF大鼠进行修复。结果显示超高速离心法可以成功分离获得外泌体，并通过电镜、粒径分析鉴定，western检测显示CD9、CD81阳性。A-MSCs外泌体原位注射POF大鼠卵巢，结果发现注射14天后，动情周期恢复正常，血液中性激素水平（E2/AMH/P）较对照组明显升高。与对照组相比，卵巢组织基因表达模式发生显著改变，KEGG分析发现A-MSCs外泌体注射组大鼠卵巢mTOR信号通路明显上调。本实验证明了A-MSCs外泌体可以对放疗导致的POF大鼠进行修复，为临床上建立一种安全有效的POF治疗新方法奠定了基础。