母-胎界面RANKL低表达诱导蜕膜COX-2+NK细胞分化异常致复发流产的机制研究

Recurrent spontaneous abortion (RSA) is associated with maternal-fetal immunomodulatory disorders, often manifested as abnormal function and phenotype of decidua immune cells (DIC). Decidua NK cells (dNK), as the highest proportion of decidua immune cells, are involved in promoting endometrial decidualization, regulating trophoblast invasion, placental vascular remodeling and maternal-fetal immune tolerance, but the specific mechanism of the differentiation and development of dNK cells has not been elucidated yet. Our previous study found that high expression of RANKL in maternal-fetal interface of normal early pregnancy could promote the differentiation of COX-2+dNK and further facilitate the invasion of trophoblast cells, while the expression of RANKL in decidua of RSA patients decreased with lower proportion of COX-2+dNK cells. The cytotoxity of uterine COX-2+NK cells of RANKL knockout mice enhanced with embryo absorption rate increased. Based on these results, we proposed a scientific hypothesis that low expression of RANKL at maternal-fetal interface induces abnormal differentiation of COX-2+dNK, leading to recurrent abortion. Therefore, taking advantage of in vitro trials for imitating the maternal-fetal microenvironment and in vivo animal experiments, our study tends to investigate the molecular mechanism of the differentiation of COX-2+dNK induced by RANKL and the pathogenesis of abnormal RSA, so as to provide a new direction for researching the mechanism of maternal-fetal immune tolerance and to explore a scientific basis for the new strategy for the diagnosis and treatment of recurrent miscarriage.

复发流产（RSA）与母胎免疫调节紊乱相关，常表现为蜕膜免疫细胞（DIC）表型与功能异常。早孕期蜕膜NK细胞（dNK）作为占比最高的DIC，参与调节子宫内膜蜕膜化、滋养细胞侵袭、胎盘血管重铸和母-胎免疫耐受等过程，但其在蜕膜局部分化发育的分子机制至今未明。我们前期研究发现人正常早孕母-胎界面高表达RANKL，可诱导COX-2+dNK分化，进而促进滋养细胞侵袭，而RSA患者蜕膜RANKL低表达，COX-2+dNK比例下降；RANKL基因敲除孕鼠子宫COX-2+NK杀伤活性增强，胚胎吸收率升高，据此我们提出科学假说“母-胎界面RANKL低表达诱导COX-2+dNK分化异常致RSA”。本研究以体外模拟母-胎界面微环境和体内动物实验为研究手段，拟阐明RANKL调节COX-2+dNK分化的分子机制及其异常致RSA的病理机制，为母-胎免疫耐受机制研究提供新方向，为探索诊治RSA的新策略提供科学依据。

成功妊娠需要母体对胚胎抗原免疫耐受，这一过程有赖于具有区域组织特异性的蜕膜免疫细胞的分化发育。一旦蜕膜免疫细胞分化异常，势必导致母体对胚胎的免疫排斥，在孕早期主要表现为反复自然流产和反复着床失败，严重危害母婴健康。已有报道显示蜕膜自然杀伤细胞(dNK)参与母-胎免疫耐受的建立和维持，然而妊娠期dNK的分化机制尚不明确。本课题以体外模拟母胎界面微环境和体内动物实验(借助WT、Tnfsf11-/-和Ptgs2-/-小鼠)为研究手段，以正常妊娠妇女和复发流产患者为研究对象， 以RANKL对蜕膜NK分化发育的调节作用为切入点。拟开展以下研究：(1)分析母-胎界面RANKL 通过何种下游信号和分子介导蜕膜 NK向COX-2+表型分化;(2)阐明蜕膜 COX-2+NK细胞调控滋养细胞生物学行为的作用机制;(3)解析RANKL 低表达介导蜕膜COX-2+NK分化异常诱导自发流产的病理机制。. 我们研究发现ESC蜕膜化后RANKL表达升高，能与NK细胞表面RANK结合，活化NK细胞内NF-κB通路，直接与PTGS2/COX-2启动子相应位点结合，促进NK细胞PTGS2转录与COX-2表达，进而上调NK细胞PGE2分泌水平，进一步抑制NK细胞转录因子IRF-1表达，促进其向耐受表型分化。而蜕膜化训导所得COX-2+CD16-NK又能高表达LDHA，代谢产生乳酸，与ESC蜕膜化后一起促进滋养细胞侵袭。因此，ESC蜕膜化在调节自身分化同时还作用于NK细胞，在母胎界面局部诱导免疫耐受的微环境。而经蜕膜化训导的NK细胞又能作用于滋养细胞，促进滋养细胞的侵袭和种植。本课题阐明了RANKL调节COX-2+dNK分化发育的作用及其机制，并进一步阐释了COX-2+dNK调节滋养细胞侵袭的相关机制，不仅对于完善正常母-胎交互对话机制和母-胎免疫耐受理论具有重要意义，而且为拓展RSA免疫发病机制研究提供了新方向，为寻找探索RSA防治新策略提供科学依据。