NETs激活血管内皮NLRP3炎症小体调控Th17细胞共同参与深静脉血栓形成的机制研究

Polymorphonuclear neutrophils (PMN) can generate and release neutrophil extracelluar traps (NETs) during inflammation. It has been found that NETs is closely related to deep vein thrombosis (DVT) in our previous studies. The specific mechanism of NLRP3 inflammasome activation in endothelial cells (EC), which could be associated with DVT, remains unknown at present. It has been demonstrated that Th17 cell may be an important mediator of PMN and EC during DVT. Therefore, we propose the hypothesis that the tissue factor can directly stimulate PMN to generate and release NETs, thereby to activate the NLRP3 inflammasome in EC by TLR9/NF-κB pathway. Then EC will secrete the mature IL-1β which activates Th17 cell to release cytokines such as IL-17 to recruit more PMNs. NETs can promote the EC to recruit, adhere, and activate platelets, and finally leading to DVT. We will detect the blood and tissue samples from DVT patients and then do the in vivo experiments using the wild-type and NETs-deficient mice and in vitro experiments using endothelial cell lines. Several experiment methods (e.g. gene knock-out, immunofluorescence stain, dual-luciferase reporter gene analysis, EMSA, ChIP, FACS) will be used in our research. The aim of this study is to clarify the mechanism of NLRP3 inflammasome activation in endothelial cell and sequent Th17 cell recruitment and excitation by NETs during DVT, which may provide new clues for the clinical treatment of DVT patients.

炎症反应时中性粒细胞（PMN）可产生胞外陷阱网结构（NETs）。我们前期研究发现NETs与深静脉血栓形成（DVT）密切相关。血管内皮细胞（EC）内NLRP3炎症小体活化可促进DVT发生，但具体机制不详。研究显示Th17细胞可能是介导PMN和EC参与DVT的重要桥梁。因此，本课题提出假说：组织因子可直接刺激PMN释放NETs，NETs通过TLR9/NF-κB通路激活EC内NLRP3炎症小体，促进IL-1β成熟及分泌，刺激Th17细胞释放IL-17招募更多PMN。NETs促进EC招募、粘附、活化血小板，最终参与DVT发生。我们将利用临床样本检测、NETs缺陷小鼠和体外细胞实验研究，使用基因敲除、免疫荧光染色、双荧光素酶报告基因分析、EMSA、ChIP、FACS等实验技术和检测方法，阐明NETs激活EC内NLRP3炎症小体调控Th17细胞共同参与DVT形成的具体机制，为DVT临床治疗提供新思路。

下肢深静脉血栓形成（DVT）是血管外科常见疾病，死亡率在心血管疾病中排名第三，但具体发病机制仍为得到充分解释。内皮细胞炎症与DVT发生关系密切，中性粒细胞（PMN）可产生胞外陷阱网结构（NETs），NETs可能调控血管内皮细胞（EC）、Th17细胞等共同参与DVT形成。本课题研究小组通过临床样本检测、NETs缺陷小鼠体内实验和体外细胞实验研究，使用基因敲除、免疫荧光染色、双荧光素酶报告基因分析、EMSA、ChIP、Western Blot、q-PCR等实验方法，证实PMN受组织因子刺激后释放NETs，通过TLR-9/NF-κB通路激活EC内NLRP3炎症小体，促进IL-1β的成熟及分泌，募集Th17细胞至炎症部位释放IL-17。IL-17招募更多PMN至反应部位形成正反馈机制。受NETs作用后，EC可对血小板进行募集、粘附和激活，血小板最终在IL-17作用下发生聚集，参与DVT发生。同时课题组依托本课题基础实验的方法和整体研究思路，围绕NETs及DVT诊治两项关键词展开系列拓展研究，就NETs实验检测方法进展、NETs在糖尿病溃疡中的具体机制、DVT及房颤患者的抗凝规范、肠系膜上静脉血栓不同累及部位患者的临床转归、髂静脉支架置入的进展等方面展开多层次研究。围绕NETs参与以DVT为主的外周血管疾病进行了丰富的探索，为阐明PMN激发EC炎症参与DVT发生的具体机制以及未来为开发新型抗血栓药物提供精确的研究窗口和潜在靶点。