滋养层细胞外泌体miR-223调控巨噬细胞功能参与URSA的机制研究

Unexplained recurrent spontaneous abortion (URSA) is a ticklish problem in refractory infertility and remains to be solved. The abnormality of maternal-fetal immune tolerance is one of the reasons of URSA, in which macrophages played an important role. During embryo implantation, trophoblast cells and decidual macrophages influence reciprocally, and macrophages contribute to the formation of maternal-fetal immune tolerance by reducing the Th1/Th2 ratio, removing apoptotic cells and decreasing antigen presentation.It has been demonstrated that the decidual macrophages of URSA patients are dysfunction,and the maternal-fetal immune tolerance is broken, nevertheless, the mechanism is not clear.Exosomal miRNAs can be carried by trophoblast cells to decidual macrophages and play its function.In previous study, we found that decidua M1-macrophages are predominate in URSA patients, miR-223 was significantly down-regulated in in trophoblast cells exosomes, macrophages showed M2 phenotype after transfection of miR-223-mimics in vitro, and bioinformatics analysis predicted that FoxO1 is the target of miR-223 in macrophages. The present study is aimed to explore the mechanism of macrophage function regulated by trophoblast cell exosomes miR-223 in URSA through in vitro and in vivo experiments covering gene, molecular, cell and individual levels.

原因不明复发性流产(URSA)是难治性不育亟待解决的问题，母胎界面免疫耐受异常是URSA重要的发病机制，巨噬细胞对母胎免疫耐受的形成具有关键的作用。胚胎植入时滋养层细胞与蜕膜巨噬细胞相互影响，巨噬细胞通过降低Th1/Th2比值、清除凋亡细胞、降低抗原提呈参与母胎免疫耐受的形成。蜕膜巨噬细胞功能异常，导致母胎免疫耐受异常，但机制未明。滋养层细胞可通过外泌体将其携带的miRNAs递送至蜕膜巨噬细胞发挥作用。前期研究我们发现URSA患者蜕膜M1型巨噬细胞占优势，滋养层细胞外泌体中miR-223显著下调，巨噬细胞体外转染miR-223-mimics呈现M2样表型改变，生物信息学预测miR-223作用于与巨噬细胞功能密切相关靶分子FoxO1。本课题拟结合体外与体内实验，从基因、分子、细胞及个体层次，探索滋养层细胞外泌体miR-223通过靶分子FoxO1调控蜕膜巨噬细胞功能导致URSA发生的机制。

巨噬细胞是一群异质性细胞,随着局部微环境的改变，其极化表型与功能不断发生改变。巨噬细胞在不同微环境极化为M1型（经典激活途径，参与杀伤病原体）和M2型（替代激活途径，抑制炎症，参与组织操作修复）。正常妊娠时，巨噬细胞在胚胎种植区域显著增多，占蜕膜免疫细胞总数的20%-30%左右，同时参与天然免疫应答和获得性免疫应答，巨噬细胞功能异常会使母胎免疫耐受发生紊乱，与原因不明复发性流产(URSA, Unexplained Recurrent Spontaneous Abortion) 等不良妊娠结局的发生密切相关。胚胎植入时滋养细胞与蜕膜巨噬细胞密切接触，通过调控其功能维持母胎免疫耐受，但机制不明。前期研究发现URSA患者滋养层细胞外泌体中miR-223显著下调。本研究检测了滋养层细胞外泌体miR-223对蜕膜巨噬细胞表面M1型和M2型膜功能分子及主要细胞因子表达水平的影响，结果表明URSA患者滋养层细胞外泌体中miR-223显著下调，在THP-1细胞中降低miR-223的表达水平，其分化成M2型细胞数明显减少；M2型标志物(Arg1，Mrc2，IL-10， CCL22等)的mRNA水平降低, 细胞上清中IL-10、 CCL22 明显降低。而增强miR-223的表达水平后，导致蜕膜巨噬细胞表面M1型膜相关功能分子（CD80、CD86）表达下调，而M2型膜相关功能分子（Arg1，Mrc2，IL-10， CCL22等）的表达水平上调，细胞上清中IL-10、 CCL22 明显升高。通过生物信息学预测及双荧光素酶实验验证了miR-223 与FoxO1的靶向关系，结果显示miR-223可以通过作用于FoxO1调控蜕膜巨噬细胞的功能。miR-223表达下调导致蜕膜巨噬细胞表型由M2型向M1型极化，使蜕膜巨噬细胞维持母胎免疫耐受的功能发生紊乱，最终可能参与了URSA的发生，我们的发现为URSA的治疗提供了新的方法策略。