hPMSCs激活mTOR通路并降低p62泛素化协同抑制自噬改善化疗源卵巢损伤的作用及机制

Chemotherapy inevitably leads to premature ovarian failure/insufficiency(POF/POI) in young women. Currently it cannot find a satisfactory control strategy to prevent chemotherapy-induced ovary damage. Human placenta-derived mesenchymal stem cells (hPMSCs)have higher expression of HGF, IGF-II and PDGF-aa than bone marrow adipose-derived MSCs. To investigate whether hPMSCs-secreting cytokines act as molecular basis to attenuate chemotherapy-induced ovarian injury, we will evaluate the ovarian function with unilateral ovary of POF/POI rats model, natural recovery group hPMSCs transplantation group and primordial follicles and mesenchymal cells co-cultivated model. It was found that hPMSCs transplantation could inhibit ovarian cell death induced by continuous autophagy. Bioinformatics analysis of RNA-seq data revealed that growth factor-mediated mTOR pathway activation and decreased ubiquitination level to inhibit autophagy as a potential mechanism. Therefore, we will investigate the differences in autophagy associated protein expression, changes in autophagy level and the association with mTOR activation and ubiquitination level of injured cells by blocking the paracrine and exosome systems of hPMSCs.We further discuss the effect and mechanism of hPMSCs on the inhibition of autophagy and homeostasis in damaged ovaries, and to clarify the protective advantages of hPMSCs in regulating the survival of functional cells, restarting the development of a pool of primordial follicles, hormone secretion and other reproductive functions.

肿瘤治疗引起化疗性卵巢早衰/功能不全(POF/POI)的发病率逐年上升，间充质干细胞防治POF/POI已成为热点，但急需明确防治效果与机制。课题组和已有报道显示胎盘间充质干细胞(PMSCs)移植可起到抗氧化和改善卵巢功能的作用，组学数据和前期预实验证实hPMSC移植可抑制化疗受损POF卵巢的过度自噬作用而提高修复效果，但机制尚未完全阐明。在以往基础上本研究拟采用环磷酰胺致POF大鼠hPMSC移植、卵巢皮质和原始卵泡与hPMSCs共培养模型，hPMSCs旁分泌和外泌体系统阻断、自噬抑制剂与双荧光mRFP-eGFP-LC3体系等方法，阐明hPMSCs对损伤卵巢中自噬抑制和稳态平衡的影响，进一步探讨hPMSCs通过mTOR激活和P62泛素化水平降低而抑制自噬的机制，明确hPMSCs在调节细胞存活、重启卵泡发育和激素分泌等生殖功能方面的保护优势，为化疗性卵巢功能衰竭患者的临床治疗提供实验基础。

化疗药物广泛应用于各种恶性肿瘤的治疗，不可避免地导致年轻女性患者卵巢的进行性损伤，导致生育能力下降。目前还没有令人满意的保护策略来避免化疗引起的卵巢功能不全。本项目研究了化疗致卵巢功能不全大鼠模型中hPMSCs移植治疗改善卵巢功能的作用。通过RNAseq差异基因分析，聚焦了NLRP3介导的炎症通路， ERK和mTOR通路等干细胞干预后的作用途径。通过本研究得到以下结果：① 化疗导致的卵巢功能不全，是卵巢细胞中PANoptosis(凋亡、焦亡和坏死)导致的细胞死亡，而细胞自噬通量的超载与死亡信号通路的串联导致了卵巢细胞功能的丧失。② hPMSCs的旁分泌蛋白谱研究结果显示粘附分子和巨噬细胞趋化因子的基因表达水平增强BMP-7、 PIGF、 IGF、 SCF、组织蛋白酶 S 和 GH的表达水平增高可促进巨噬细胞增殖和 M2极化。CEACAM-1，ICAM-1，IP-10，I-TAC，RANTES 和 NT-3，降低了卵巢纤维化水平，起到抗氧化应激作用，进而修复受损卵巢、改善卵巢功能。胎盘间充质干细胞通过激活ERK、mTOR通路、抑制NLRP3炎症小体通路，降低了损伤细胞中非选择自噬的过度激活。③胎盘间充质干细胞通过降低总泛素化水平，抑制p62泛素化减轻自噬流受损，维持了线粒体的数量，增加了卵巢细胞的氧化磷酸化代谢功能。本研究为阐明MSCs改善化疗所致卵巢损伤的机制开辟路径，为MSCs治疗POI的临床转化提供理论依据。