妊娠早期母猪血液外泌体中高表达的miR-192在胚胎附植中的调控机制研究

miR-192在妊娠14天的母猪血液外泌体中高表达且其表达量极显著高于配种未妊娠的母猪，并有很多预测的miR-192靶基因与胚胎附植有关，这提示miR-192参与胚胎附植过程。但妊娠早期母猪血液外泌体中的miR-192来源、作用的靶基因及调控机制都不清楚。本项目拟采集配种后第14天妊娠母猪和对照母猪子宫内膜及其他组织样，通过iTRAQ鉴定在母猪胚胎附植点子宫内膜显著下调的蛋白，筛选miR-192靶基因并构建双荧光素酶报告载体验证；通过qRT-PCR试验判断血液外泌体miR-192的可能来源；通过免疫组化确定靶基因的定位与表达规律；在滋养层细胞、子宫内膜上皮细胞和活体早孕模型小鼠子宫角中使用miR-192拟似物和抑制剂分析其调控作用。本研究将首次开展母体血液外泌体miRNA调控胚胎附植的研究，系统地揭示miR-192在猪胚胎附植中的调控作用机制，为深入理解妊娠早期母胎通讯机制提供新的视野。

The exosome miRNAs in peripheral blood could be a kind of ideal biomarker for diagnosis. In previous research the proposer found that: miRNA-192 was highly expressed in serum exosome of sows at day 14 of pregnancy, and was higher than that of sows unpregnant very significantly, and many target genes predicted involved in embyo development and implantation. The results shown that miRNA-192 participate in embryo implantation at early pregnancy stage, but where does miRNA-192 of blood exosome come from, where does it go, what are its target genes and how does it regulate embryo implantation, are all unkown yet..In this proposal, early embryos, endometrium samples and other tissues of sows pregnant and unpregnant will be collected; endometrium at embryo attachment site of sows pregnant and endometrium of control sows will be used to conduct iTRAQ to screen proteins differentially expressed, target genes of miR-192 will be selected combined with bioinformatics tools and verified by using dual-luciferase reporter system. The source of miR-192 in serum exosome will be determined by quantitative expression analysis, and the expression location of target genes in placenta and embryo of early pregnancy will be determined by immunohistochemistry. The regulation of miR-192 on trophoblastic cells and endometrial epithelial cells will be analyzed in vitro, the mimics and inhibitor of miR-192 will be injected into uterine horn of mice pregnant to detect the regulation of miR-192. This study will reveal the role of miR-192 in the process of embryo-inplantation, and will provide a new vision for further understanding the mechanism of pregnancy regulation.