Pg-LPS刺激血小板释放微粒对单核细胞作用及促动脉粥样硬化的机制研究

感染是动脉粥样硬化（AS）的一个重要发病因素，牙周感染促AS发生和发展是研究热点。P.gingivalis和Pg-LPS可诱导血小板活化，不同刺激和病理状态下血小板活化释放微粒（PMPs）特征和功能有差异。推测Pg/LPS刺激血小板活化释放携带母体特征的PMPs，扩大信号传递和调节炎症细胞功能，流动至远隔部位参与AS过程。本研究采用流式细胞术，RT-PCR，Western Blot等方法，分析LPS-PMPs膜糖蛋白表达及通路，其对单核细胞迁移、炎症因子分泌、CD16+亚型表达及巨噬细胞向泡沫细胞分化的作用及机制，干扰CD40-CD40L和P-selectin-PSGL-1结合对其作用的影响。在小鼠AS和牙周炎模型中分析牙周炎症与PMPs的动态关系和LPS-PMPs促动脉斑块形成和稳定性的作用。研究结果从理论上丰富牙周炎对全身健康的影响，为临床治疗提供理论依据和治疗靶点。

Inflammation has an important role in the pathology of atherosclerosis. Periodontitis are kinds of infectious inflammatory diseases, which may promote the formation and development of atherosclerosis, but there is still lack of direct evidence of the association Porphyromonas gingivalis is a major etiological agent of periodontal diseases, the virulence of which has been attributed to different factors, including lipopolysaccharide (LPS). From literatures and our previous studies, it was demonstrated that platelet activation is high in patients with periodontitis. Platelet CD62P expression, and platelet reactivation to P. gingivalis increased in patients with periodontitis. P. gingivalis and Pg-LPS can stimulate platelet activation and promote the formation of platelet and leukocyte aggregates. Platelets have the propensity to release microparticles (PMPs) upon activation, which may mediate inflammatory responses and contribute to exacerbate inflammatory diseases and conditions. Carrying components of the platelets' cytoplasm, PMPs may exert their effects on recipient cells by transferring their content in platelet-derived bioactive mediators, cytokines, mRNAs and microRNAs. The available evidence indicates that shedding of microparticles from the parental cells is not just a passive process accompanying cellular dysfunction and apoptosis, but a tightly regulated mechanism implicated in the interactions between various cell types. The functions platelets are achieved by a variety of surface receptors, and the receptor were also expressed on the PMPs, contribute to cross-talk with inflammatory cells. Our hypothesis is that in periodontitis, the activated platelet stimulated by periodontal pathogens shed platelet microparticles (PMPs), which maybe different with microparticles shedding from apoptosis platelet, PMPs transfers the receptors and inflammation signals of periodontitis to remote organs and magnifies the periodontal inflammatory effect by regulating the function of other immune inflammatory cells, and may play a role in the formation and development of atherosclerosis. The present study will focus on analysis of platelet microparticles stimulated by P. gingivalis or LPS (LPS-PMPs), its characteristics and role in the connection between periodontitis and atherosclerosis will be elaborated. Confocal microscopes and scanning electron microscopes will be used to reveal the morphology of LPS-PMPs and aggregates with monocytes. The membrane glycoprotein expression of GPIIb，CD41a，CD62P on PMPs will be analyzed by high sensitivity flow cytometry. Transwell chambers, RT-PCR, Western blot, immunohistochemistry, and ELISA will be used to analyze the roles and mechanisms of LPS-PMPs on the migration, inflammatory cytokines secretion of monocytes, the effect of block P-selectin-PSGL-1 and CD 40L-CD 40 binding in cell signals of the interaction will be analyzed. The roles of LPS-PMPs in promoting THP-1 origin macrophage cells to differentiate into foamy cells will be unravelling. Animal models of experimental atherosclerosis and experimental periodontitis in ApoE-/- mice and C57BL/6 mice will be used to analyze the dynamic effect of periodontal destruction on PMPs shedding and membrane glycoprotein expression, and LPS-PMPs in the morphology and stability of carotid artery plaque in vivo will be analyzed. The present study in LPS-PMPs will provide a new appreciation of the role of periodontitis in atherosclerosis, and apply some new data to elucidate mechanisms as well as enrich the theory of periodontitis on atherosclerosis, unravelling the details of inflammatory pathways may eventually furnish new therapeutic targets, and give more clues for understanding the clinical benefits of periodontal therapies.