含HLA-G外泌体通过影响蜕膜NK细胞功能参与早期复发性流产的机制研究

The interaction between trophoblast HLA-G and decidual NK (dNK) cells on maternal-fetal interface plays a vital role in the maintance of early-term gestation, and the dysregulation of both can cause recurrent spontaneous abortion (RSA). Our previous study showed that decrease of HLA-G induced by miR-133a in trophoblast cells can disturb the proinvasion and proangiogenesis potential of dNK cells, which may be an important cause of RSA. However, the specific mechanism of down-regulation of HLA-G-induced dysfunction of dNK cells remains unclear. HLA-G-expressing exosomes which can exist in maternal blood and maternal-fetal interface are important to the normal pregnancy. Therefore, we hypothesize that abnormal secretion of HLA-G-expressing exosomes derived from maternal-fetal interface and peripheral blood of RSA patients interferes with the function of dNK cells and disturbs trophoblast invasion and uterine spiral arteries remodeling, then finally leads to RSA. On the basis of previous study, we will use early abortion chorionic villi; maternal decidua and peripheral blood samples; RSA mouse models as materials, by adopting cytology and immunology techniques to explore the mechanism that HLA-G-expressing exosomes are involved in early-term RSA. This study aims to reveal the new mechanism of RSA occurrence at the molecular and cellular levels, which is expected to provide targets and strategies for the clinical precise prevention and treatment of RSA.

存在于母胎界面胚胎滋养细胞的HLA-G与母体蜕膜NK细胞之间的相互作用对于维持早期妊娠具有重要作用，二者功能失调会导致复发性流产（RSA）。本课题组前期研究发现，miR-133a可负调控HLA-G的表达，HLA-G下调使蜕膜NK细胞促侵袭和促血管生成功能受损，这可能是RSA发生的重要原因。但下调的HLA-G引起蜕膜NK细胞功能失调的具体机制尚未阐明。含HLA-G外泌体可存在于胎盘和孕妇外周血中，对维持正常妊娠具有重要作用；因此，我们提出假说：RSA孕妇母胎界面和外周血含HLA-G的外泌体分泌异常，影响了蜕膜NK细胞功能，导致RSA发生。拟在前期研究基础上，选用孕早期流产绒毛组织、母体蜕膜组织及外周血样本、RSA小鼠模型等为材料，通过细胞学、免疫学等方法探索含HLA-G外泌体参与早期RSA的机制。本研究以期在分子与细胞层面揭示RSA发生的新机制，可望为临床精准预防和治疗RSA提供靶点及策略。

本研究严格按照申请书的研究内容执行。首先，我们通过收集并提取人工流产（Induced abortion， IA）与不明原因复发性流产（Unexplained recurrent pregnancy loss, uRPL）患者外周血血清外泌体分析发现，与正常妊娠和偶发性流产（Sporadic pregnancy loss, SPL）相比，uRPL患者血清外泌体miRNAs表达谱存在显著差异，对差异miRNAs进行进一步验证并筛选出差异最为显著并可能参与uRPL发生的miRNA：miR-185-5p作为预测分子标志物进行进一步验证。结果发现，血清外泌体miR-185-5p可以较好的预测uRPL患者再次流产的发生，是uRPL患者的潜在预测标志物。.其次通过收集IA患者绒毛组织外泌体并与蜕膜NK细胞（dNK）共培养发现，IA-EXO中含有高浓度的HLA-G，dNK细胞可大量摄取外泌体，并可显著降低dNK细胞促炎因子IL-8和IFN-γ的表达水平，增强dNK细胞VEGF和促生长因子OGN、PTN的表达水平；再次通过收集IA与uRPL患者绒毛组织外泌体发现，与IA患者相比，uRPL患者绒毛组织外泌体（EXO）中HLA-G含量明显下降，促dNK细胞分泌促生长因子功能下降；同时在滋养细胞细胞系HTR-8外泌体中过表达HLA-G可以显著增强其促生长因子功能；进一步对两组外泌体进行miRNA测序分析发现，uRPL-EXO中miR-29家族表达水平显著下调；通过人类临床样本发现，外泌体中miR-29a的表达水平与其抑炎功能呈正相关；通过双荧光素酶试验证实，miR-29a与IFN-γ 3’ UTR区存在直接结合位点，并且通过向外泌体中直接电转染miR-29a 抑制剂可显著抑制其抑炎功能，提示IA-EXO可能通过miR-29a调控dNK细胞IFN-γ的表达水平。体外治疗实验发现，在IA-EXO内负载miR-29a模拟物可显著改善uRPL患者dNK细胞的高炎症状态，并可改善dNK细胞的促血管生成能力；通过构建可缓释外泌体的宫腔内给药系统，发现IA-EXO内负载miR-29a模拟物可显著改善uRPL小鼠模型的炎症水平，降低小鼠的流产率。本研究按着项目计划书（81871182）执行，完成了既定的研究内容。