子宫内膜上皮细胞源外泌体bta-miR-218调控子宫内膜炎奶牛胚胎滋养层细胞植入功能的机制研究

Maternal-fetal dialogue adjustment disorder caused by chronic endometritis is the main reason of causing cow reproduction obstruction. Endometrial epithelial cells derived exosomes miRNA play important roles in reproductive function of animals and early embryo development. Revealling the impact and mechanism of endometrial epithelial cells derived exosomes miRNA on trophoblast cell will provide new theoretical foundation and basis for a comprehensive understanding of maternal-fetal dialog and cows infertility prevention and control. Our previous study found that bta-miR-218 is a high expression miRNA in endometrial epithelial cell derived exosomes under endometrial inflammation conditions. However, the specific regulation mechanism of bta-miR-218 on trophoblast cell implantation function is still unknown. In this project, in situ hybridization, qRT-PCR and immunofluorescence methods were used to firstly clarify the bta-miR-218 differential expression in endometritis cows uterus and its transmission rule from endometrial epithelial cells to trophoblast cells. After that, endometrial stereoscopic/3D model and gene silence/overexpression techniques were used to further explore the molecular mechanisms of bta-miR-218 and its downstream target genes on bovine trophoblast cell proliferation, apoptosis, migration and invasion. Thus illustrating the mechanism of dairy cow embryo implantation influenced by endometritis from a new perspective.

慢性子宫内膜炎引起的母-胎对话调节紊乱是造成奶牛繁殖障碍的主要原因。子宫内膜上皮细胞源外泌体（exosomes）及其包含miRNA在动物生殖功能发挥和胚胎早期发育等过程中具有重要作用。揭示其对胚胎滋养层细胞的影响及其机制可为全面认识母-胎对话的调控和奶牛不孕防治提供新的理论基础和依据。前期研究发现，bta-miR-218是子宫内膜炎症条件下牛子宫内膜上皮细胞来源外泌体中一个高表达的miRNA，其具体调控胚胎滋养层细胞功能的机制尚不清楚。本项目通过原位杂交、qRT-PCR、免疫荧光等方法明确bta-miR-218在子宫内膜炎奶牛子宫中表达及随外泌体在子宫内膜细胞-胚胎滋养层细胞间传递规律；通过细胞3D培养模型和基因沉默/过表达技术，进一步阐明bta-miR-218及其调节的下游靶基因影响胚胎滋养层细胞增殖、凋亡、迁移力和侵袭力的分子机制，进而从新的角度揭示子宫内膜炎影响奶牛胚胎附植的机制。

子宫内膜细胞损伤和慢性炎症是造成奶牛繁殖障碍的主要原因。子宫内膜上皮细胞源外泌体（exosomes）及其特异性miRNA在动物生殖功能发挥、子宫局部炎症发生和母-胎间精确对话建立等过程中具有重要作用。深入揭示子宫内膜上皮细胞源外泌体特异性miRNA在子宫内膜细胞炎症中的调控机制，对于子宫内膜炎的防控、提高妊娠率、保证胎儿的健康具有重要意义。在前期研究发现，bta-miR-218在子宫内膜炎症条件下是子宫内膜上皮细胞来源外泌体中特异表达的一个miRNA。本项目通过细胞3D培养模型和胚胎共培养技术，进一步探索子宫内膜炎症条件下牛子宫内膜上皮细胞源外泌体bta-miR-218及其调节的下游靶基因对牛胚胎体外发育能力、子宫内膜容受性相关因子Galectin15、GlyCAM-1、MUC-1、ISG15表达的影响。在本项目中，我们首先鉴定了奶牛阴道菌群的多样性与产后子宫内膜炎的相关性，利用大肠杆菌脂多糖构建了奶牛子宫内膜炎体外模型，成功构建了奶牛胎盘滋养层细胞系；从奶牛子宫腔液、乳汁、子宫内膜上皮细胞、乳腺上皮细胞和胎盘滋养层细胞中稳定分离得到了外泌体囊泡。随后我们进一步发现奶牛子宫内膜上皮细胞源外泌体miR-218与子宫内膜炎子宫和细胞炎性状态有关；外泌体来源miR-218能够通过外泌体与邻近子宫内膜上皮细胞的融合调节子宫内膜上皮细胞内免疫因子和趋化因子的释放；外泌体及其来源miR-218能够靶向sFRP2调节早期胚胎的发育和奶牛早期胎盘的形成；LPS刺激子宫内膜上皮细胞来源外泌体能够激活CD4+和CD8+T细胞，其激活模式与产后21 d以后患子宫内膜炎奶牛子宫局部T淋巴细胞的变化趋势一致。本项目为外泌体miR-218作为奶牛子宫内膜炎防控新靶点提供了实验依据，进一步阐明了子宫内膜炎引起奶牛繁殖障碍的新机制。