中性粒细胞胞外诱捕网（NETs）在抗磷脂抗体介导的胎盘源性病理妊娠中的作用机制

The antiphospholipid antibodies play important roles in thrombosis diseases and placenta-mediated pregnancy complications such as recurrent spontaneous abortion,pre-eclampsia,fetal growth restriction,stillbirth.However,the machenism is unexplained.Recently,the role of neutrophil extracellular traps(NETs) in antiphospholipid antibody-mediated thrombosis attracts more and more researchers,who have revealed that NETs caused directly damages to epithelial and endothelial cells.What,s more,a more robust non-infectious infiltrate of both intact neutrophils and NETs in the placental intervillous space was present in both lupus and pre-eclampsia compared with control pregnancies.Up to now,there is nobody investigating the role of NETs in placenta-mediated pregnancy complications.This subject aims to understand the ability of neutrophils in peripheral blood and placenta in pregnant women with antiphosphlipid antibodies and the machenism of NET release.We are also intrested in impacts of NETs in trophoblasts and how will aspirin and low molecular weight heparin interact with them.Maybe,the same NET contents that disarm and kill bacteria extracellularly are toxic to endothelial cells in vitro and have similar toxic or pro-apopototic effects on trophoblasts,resulting in impaired placentation.The main research technologies we will used are immunofluorescence,Western blot,flow-cytometry,et al.We will use a cofocal microscopy and a electron microscopy to observe the NETs.Understanding the molecular pathways leading to NETs generation after neutrophil activation will give us a better insight into the pathogenesis of placenta-mediated pregnancy complications induced by antiphospholipid antibodies and might be useful for the therapeutics.

抗磷脂抗体（aPLs）与复发性流产、子痫前期、胎儿生长受限等胎盘源性病理妊娠有关，而其导致这些病理妊娠的发病机制尚不明确。近年来研究发现中性粒细胞胞外诱捕网（NETs）在抗磷脂综合征血栓形成中有重要作用。在子痫前期、SLE妊娠胎盘中，NETs浸润明显增加，而NETs在aPLs阳性的病理妊娠中作用机制如何，国内外尚未见报道。本课题拟通过体内、体外实验，利用免疫荧光、细胞共培养、Western blot、流式细胞分析等技术，使用激光共聚焦显微镜、电镜等成像设备，从整体、细胞、分子三个水平研究1）aPLs阳性妊娠妇女外周血及胎盘中性粒细胞形成NETs及血浆降解NETs的能力；2）aPLs是否通过MPO-NE途径依赖PAD4刺激NETs形成；3）NETs对滋养细胞生物学行为的影响及药物干预的效果，为阐明aPLs介导的胎盘源性病理妊娠发病机制提供理论依据，以期对aPLs阳性妊娠期治疗提供新靶点。

项目背景：目前对于产科抗磷脂综合征(Obstetric antiphospholipid syndrome, OAPS)常规治疗方法为低剂量阿司匹林联合预防剂量低分子肝素治疗，80%以上 OAPS 患者可以成功妊娠，但仍有部分患者妊娠期间出现妊娠并发症，提示 OAPS 发病机制需要进一步探索。中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)作为中性粒细胞活化发挥功能的方式之一，在多种自身免疫性疾病发病过程中具有重要作用，但在OAPS发病机制中的作用机制尚不明确。.主要研究内容：.1.OAPS对孕妇体内NETs水平的影响；.2.产科抗磷脂综合征患者体内抗磷脂抗体对NETs生成的促进作用及其机制；.3.抗磷脂抗体诱导的NETs对滋养细胞及内皮细胞功能的影响。.重要结果：.1.早孕期产科抗磷脂综合征患者体内NETs 水平升高，其中性粒细胞具有 NETs形成倾向。.2.早孕期产科抗磷脂综合征患者血清中纯化的IgG可以刺激正常妊娠者中性粒细胞释放胞外诱捕网；APS-IgG诱导NETs生成依赖ROS生成及Akt、ERK及p38-MAPK磷酸化。.3.APS-IgG诱导生成的NETs抑制滋养细胞的侵袭及迁移能力，对迁移能力的抑制作用可被DNase I部分拮抗；APS-IgG诱导生成的NETs抑制内皮细胞的迁移及血管生成功能，对血管生成功能的抑制作用可被DNase I部分拮抗。.关键数据：.1.OAPS 患者血清中游离dsDNA水平、MPO-DNA复合物含量、MPO、NE含量均明显高于正常妊娠者(P<0.05)。.2.分别用 APS-IgG、HC-IgG及PMA孵育正常妊娠组中性粒细胞，发现与HC-IgG 组相比，APS-IgG 组中性粒细胞释放游离 DNA 水平增加(P<0.05)。.3.与NT组相比，NETs组发生侵袭的滋养细胞数量明显减少(P<0.01)。成管实验结果表明，与NT组相比，NETs组内皮细胞生成小管的分支数目、节点数目、网眼数目及网眼面积均明显减少，但是当用APS-IgG诱导生成的NETs与 DNase I同时处理内皮细胞时，这种抑制作用则得到部分改善。.科学意义：初次探索OAPS孕妇早孕期体内NETs水平，揭示抗磷脂抗体诱导NETs生成机制，初探抗磷脂抗体诱导的NETs对滋养细胞及内皮细胞功能的影响。