胎盘外泌体miRNA对PD-1/PD-L1介导的奶牛妊娠免疫耐受的调控机制

Reproductive disorder is one of the most important problems restricting the development of dairy industry, and its main cause is the embryo loss in the first trimester. Disorder of maternal pregnancy immune tolerance results in embryonic loss, but its mechanism remains to be elucidated. According to our early results, this project will isolate and identify the placental exosomes from peripheral blood in pregnant cows, and its miRNAs will be deeply sequenced with Illumina genome analysis system. After that, specific miRNAs derived from placenta will be analyzed and predicted for their target mRNAs and possible molecular regulation networks. The trophoblast cells and endometrial epithelial cells will be used to confirm these special miRNAs/mRNAs and their regulating networks on pregnancy immune tolerance mediated via PD-1/PD-L1 signal pathways in vitro. These PD-1/PD-L1 networks and Th1 / Th2 cytokine levels regulated by miRNAs/mRNAs will be verified in macrophages. These networks and signal pathway will be further validated in mice in vivo. Finally, the function of special miRNAs from placental exosomes will be corroborated in pregnant dairy cows. These results could strongly suggested the molecular mechanism of special miRNAs in placental exosome regulating the pregnancy immune tolerance mediated by PD-1/PD-L1 signal pathway in dairy cows.

繁殖障碍是制约奶牛产业发展的重要问题之一。早期胚胎流失造成繁殖障碍，其原因主要是母体妊娠免疫耐受的失败，但其作用机制有待进一步阐明。本项目基于前期研究基础，分离和鉴定孕牛外周血液中胎盘外泌体，用Illumina基因组分析系统深度测序胎盘外泌体miRNAs，重点分析胎盘外泌体特异的miRNAs/mRNAs，预测其分子调控网络；采用滋养层细胞和子宫内膜腔上皮细胞确证miRNAs及其对PD-1/PD-L1信号通路的调控作用；利用巨噬细胞研究胎盘外泌体miRNAs/mRNAs对PD-1/PD-L1介导的Th1/Th2细胞因子的调节作用；小鼠模型验证miRNAs/mRNAs对PD-1/PD-L1信号通路介导妊娠免疫耐受的调控能力和机制；妊娠奶牛验证胎盘外泌体特异miRNAs对妊娠免疫耐受的调控作用。项目的完成将阐明胎盘外泌体miRNAs/mRNAs对PD-1/PD-L1介导的妊娠免疫耐受的调控机制。

胎盘外泌体在妊娠期间发挥着重要作用，胎盘外泌体miRNA 在各个妊娠期间外周血液中高量存在，可参与母胎免疫耐受机制。miRNA可调节PD-1/PD-L1信号转导通路介导的机体免疫，可能与妊娠免疫耐受机制的分子机制有关。本项目通过采用高通量测序技术分析奶牛妊娠早期、中期及晚期外周血液外泌体中差异表达的miRNAs，以探讨奶牛妊娠期间不同miRNA对免疫耐受的调控机制。同时，该项目随后也探讨了miRNA对PD-1/PD-L1的调节作用，进一步阐明了胎盘外泌体miRNA在妊娠早期子宫内膜免疫反应中的关键调节机制。.结论：（1）通过高通量测序筛选奶牛胎盘外泌体差异表达的miRNAs，GO分析发现差异表达miRNAs主要富集在蛋白结合、小分子结合等，KEEG分析发现主要富集在囊泡运输中的SNARE相互作用、Ras信号通路等。并对不同时期差异miRNAs的表达丰度进行验证。（2）通过研究胎盘外泌体miRNA对奶牛妊娠免疫耐受调控，发现妊娠早期胎盘外泌体bta-miR-499显著高表达，通过靶向Lin28B抑制NF-κB信号的激活。同时，Lin28B分子通过上调let-7 miRNAs可抑制子宫局部炎性反应。（3）探讨胎盘外泌体miRNAs对PD-1/PD-L1的调控，结果表明，PD-1/PD-L1参与母胎界面免疫微环境的调控，延缓免疫排斥。miRNAs在妊娠不同阶段通过靶向PD-L1参与妊娠免疫耐受的调控。（4）HoxA11对PD-L1表达的具有调控作用，HoxA11通过调控PD-L1的转录促进奶牛妊娠早期子宫内膜上皮细胞的重塑。.本项目诠释了奶牛母体对胚胎妊娠免疫耐受的分子机制，为奶牛母胎妊娠免疫耐受的药物研发和分子介入调节提供科学依据。