猪DIRAS3基因调控胚胎附植中子宫内膜上皮-间质转化的分子机理研究

上皮-间质转化能改变猪子宫内膜容受性进而影响胚胎附植，申请人前期研究结果表明肿瘤抑制基因DIRAS3在胚胎附植不同时期差异表达，可能是调控上皮-间质转化重要基因之一，但有关其功能和具体作用机制在猪中尚未见报道。本研究拟以猪DIRAS3为研究素材，在检测附植前后子宫内膜组织形态变化及DIRAS3表达和定位变化基础上，通过慢病毒转染介导DIRAS3基因在猪子宫内膜上皮细胞中过表达和抑制表达并检测相关信号蛋白表达，从而揭示DIRAS3基因表达量对上皮-间质转化的影响及激活的信号通路。同时，通过在附植前小鼠子宫角中注射DIRAS3抗体，研究阻断内源DIRAS3蛋白的表达对上皮细胞表型和附植的影响，从而阐明DIRAS3基因对上皮-间质转化的调控机理及其在附植中的作用。本研究将有助于对子宫内膜容受性获得的调控机理认识，并为产仔数性状的遗传改良研究提供新的基因素材，也将为探讨附植的调控研究开辟新的视角。

Epithelial-mesenchymal transition (EMT) invovled in the implantation by altering uterine receptivity, but the molecular mechanism remains elusive. Present studies established the temporal and spatial pattern of endometrial DIRAS3 expression during the peri-implantation and post-implantation stage to determine the association of DIRAS3 expression with endometrial cell morphology in pregnant gilts. To determine whether DIRAS3 is involved in EMT and found signaling pathways proposed to EMT, lentiviral-mediated transfer of DIRAS3 to primary porcine endometrial epithelial cells. Our analyses would focus on the view of the cell morphology and the mRNA and protein expression levels of gene marker specific for epithelial cells and mesenchymal cells. To verify the impact of DIRAS3 gene mediated EMT in embryo implantation, intraperitoneal (i.p.) injections with anti-DIRAS3 antibody D1 = day of vaginal plug detection) and D4 to examine the ultrastructure of the potential implantation site in female mice compared to that of wild type animals. We also compared expression of mRNA and proteins of gene marker specific for epithelial cells and mesenchymal cells. Our study would clarify the molecular mechanism of EMT mediated by DIRAS3 during the implantation and the role of DIRAS3 expression in endometriun receptivity. The research could contribute to better understanding of molecular mechanism of endometriun receptivity and provide a novel inight on the resarch about the molecular mechanism of endometrial receptivity.