miR-152靶向调控HLA-G影响蜕膜NK细胞功能参与子痫前期发病的机制研究及其胎盘外泌体源性检测

The etiology of preeclampsia (PE) is not yet well-known, and the placental factor is the main etiology theory. Our previous studies and related literature showed that: miR-152 is associated with PE, and it could assist in disease prediction; its target gene human leukocyte antigen-G (HLA-G) could affect the function of decidual natural killer cells (dNKs). In the maternal-fetal interface, dNK affect trophoblast function and vascular remodeling by secretion of relevant cytokines, and maintain the normal pregnancy. It is worth to explore whether miR-152 could participate in the process of dNK affects the function of trophoblasts through be mediated by its target gene, and whether it has exosomes origin.On the basis of the previous study, this project intends to construct a co-culture system of trophoblast cells transfected with miR-152 and dNK, and to detect the secretion function of dNK after intervention of relevant pathways as well as the effect of co-culture supernatant on trophoblast invasion and angiogenesis; what’s more, to detect the exosomes origin of miR-152. To explore whether miR-152 could affect the function of dNK cells through regulation of HLA-G, and participate in the pathogenesis of PE; additionally, whether miR-152 has the potential to become a biomarker of PE.

子痫前期（preeclampsia, PE）病因不明，胎盘因素为主要学说。我们前期研究和相关文献表明：miR-152与PE相关，可协助疾病预测；其靶基因人类白细胞抗原G（human leukocyte Antigen-G，HLA-G）可影响蜕膜 NK 细胞（decidual natural killer cells, dNK）功能。母胎界面中，dNK分泌相关因子，影响滋养细胞功能及血管重塑，维系正常妊娠。miR-152能否通过靶基因介导，参与dNK影响滋养细胞功能，且具有胎盘外泌体源性，值得探究。该项目拟构建转染miR-152的滋养细胞与dNK共培养体系，干预相关通路后检测dNK分泌功能，及共培养上清对滋养细胞侵袭、血管形成过程的影响；并对miR-152进行外泌体源性检测；探讨其能否通过对HLA-G的调控影响dNK细胞功能，进而参与PE发病的相关机制，及其是否具有成为PE疾病标志物的潜力。

子痫前期（PE）是围产期导致孕产妇及胎儿死亡的常见疾病，其病因尚不明确。微小RNA 152（miR-152）在PE患者孕晚期胎盘组织中异常表达，其与PE发病相关。本项目，从母胎界面中免疫微环境角度，进一步探究miR-152的相关功能，明确该基因调节滋养细胞和人脐静脉内皮细胞功能的具体方式及机制。首先，我们利用人早孕蜕膜组织获得了纯度较高的蜕膜自然杀伤细胞（dNKs），构建了dNKs与HTR-8细胞共培养体系。发现在HTR-8细胞中，miR-152能够在mRNA及蛋白水平负向调控HLA-G。其次，发现在dNKs表面KIR2DL4受体被阻断情况下，dNKs与转染miR-152模拟物的HTR-8共培养上清液中IL-8、IL-10、IP-10、TGF-β、VEGF、GM-CSF、LIF的水平显著降低；且该组共培养上清液可明显抑制HTR-8的侵袭能力和HUVEC成管能力，并抑制HTR-8中MMP-2、MMP-9表达。再次，发现在合理浓度干预下，GM-CSF、LIF均可促进HTR-8侵袭能力、HUVEC细胞成管能力，且增加HTR-8细胞中MMP-2和MMP-9，及HUVEC中VEGF的表达。最后，发现PE患者血清及血清外泌体中miR-152表达均增高；PE患者及正常晚孕患者血清外泌体具有典型外泌体形态及良好粒径，且均可显著表达PLAP；HTR-8能够摄取外泌体至细胞内；摄取PE患者血清外泌体之后，HTR-8细胞凋亡率增加，增殖及侵袭能力显著下降。我们的研究表明：miR-152可负性靶向调控HLA-G，在mRNA及蛋白水平均可发挥调节作用。高表达的miR-152通过HLA-G/KIR2DL4途径，抑制dNKs分泌细胞因子，进而抑制HTR-8侵袭能力和HUVEC成管能力，从母胎界面的层面参与PE发病；其中GM-CSF、LIF均在影响上述细胞相关功能方面发挥重要作用。PE患者血清中、血清外泌体中miR-152表达趋势与PE胎盘组织中一致，含miR-152外泌体具有胎盘源性；PE患者血清外泌体可抑制HTR-8细胞增殖及侵袭，并增加细胞凋亡；miR-152可能是以外泌体携带的方式在胎盘-母体间发挥作用。本项目的完成，为寻找PE病因及疾病治疗靶标提供新的思路和视角。