3D培养的间充质干细胞外泌体通过miR-25修复薄型子宫内膜的分子机制研究

Thin endometrium usually results to infertility and abortion. Mesenchymal stem cells (MSC) can repair endometrial injury.The exosomal miRNAs play an important role in regulating the repair. Many studies have shown that miR-25 repairs cells injury via PTEN/ PI3K/AKT signaling pathway.Our previous studies showed that exosome derived from MSC of 3D culture inhibited the apoptosis of endometrial cells. Compared with 2D culture, miR-25 expression derived from MSC of 3D culture significantly increases. Therefore, we hypothesize that exosomal miR-25 derived from MSC of 3D culture regulates the repair of endometrial injury through the PTEN/PI3K/AKT signaling pathway. We plans to research the mechanism in cell and animal level by microencapsulation, RT-PCR, etc. (1) MSC of 3D culture repairs endometrial injury through exosomes, inhibits apoptosis and promotes angiogenic factor expression; (2) exosomal miR-25 derived from MSC of 3D culture regulates the repairing endometrial injury through PTEN/PI3K/AKT signaling pathway. By studying the molecular mechanism of exosomal miR-25 in thin endometrium repairing, we could provide a new method for the repair of endometrial damage and treatment infertility in clinic.

薄型子宫内膜是不孕不育及流产的常见原因。间充质干细胞（MSC）可以修复薄型子宫内膜的损伤，外泌体的miRNA在其中具有重要的调控作用，研究表明miR-25可以通过PTEN/PI3K/AKT信号通路修复细胞损伤。我们前期研究结果表明3D培养MSC外泌体抑制子宫内膜细胞凋亡，而且与2D培养相比，3D培养MSC外泌体中miR-25表达显著提高。因此我们提出假设：3D培养MSC外泌体通过miR-25调控PTEN/PI3K/AKT信号通路修复薄型子宫内膜损伤。本项目拟通过微胶囊封装、RT-PCR等实验技术，在细胞和动物水平研究:(1)3D培养MSC是通过外泌体抑制子宫内膜细胞凋亡，促进VEGF表达，修复子宫内膜细胞损伤；(2)3D培养MSC外泌体通过miR-25调控PTEN/PI3K/AKT信号通路修复子宫内膜损伤。通过其分子机制研究为3D培养MSC应用于临床提供依据，为治疗不孕不育提供新方法。

薄型子宫内膜是导致不孕不育及流产的重要原因，抑制子宫内膜细胞的凋亡、促进血管再生是修复子宫内膜的损伤的关键。间充质干细胞单独移植难以达到预期的治疗效果， 3D培养技术提高干细胞活力可能是子宫内膜损伤的新方法。本项目水凝胶微胶囊封装干细胞构建3D培养干细胞（3D-MSCs）体系并进行表征，通过间接共培养发现3D培养干细胞修复子宫内膜基质细胞损伤的效果显著优于2D培养干细胞，证实3D培养干细胞通过外泌体3D-MSCs(3D-MSC-Exos）抑制损伤子宫内膜细胞的凋亡;提取3D-MSC-Exos并进行表征，证实微胶囊封装干细胞进行3D培养增加外泌体的分泌的浓度，增加外泌体修复损伤子宫内膜细胞的效果；通过3D培养与2D培养外泌体miRNA测序分析，检测3D-MSC-Exos表达显著的miRNA，并分析表达差异的miRNA促进细胞增殖，抑制细胞凋亡的效果;通过3D-MSCs和3D-MSC-Exos对于小鼠薄型子宫内膜模型的治疗效果，证实3D培养干细胞来源外泌体通过PTEN/AKT信号通路调控促进子宫内膜修复，促进血管再生；最后我们验证干细胞来源外泌体中miR-21和miR-25抑制PTEN表达，促进AKT磷酸化抗凋亡修复内膜损伤的机制。综上所述，本项目证实水凝胶微胶囊封装干细胞通过外泌体调控PTEN/AKT信号通路抑制细胞凋亡，促进血管再生，3D培养干细胞及其外泌体修复子宫内膜显著优于2D培养干细胞，为3D培养MSC应用于临床提供依据，为治疗不孕不育提供新方法。