血小板通过CD40L-CD40激活单核细胞MAPK/ERK信号通路参与脓毒症炎症反应机制研究

Monocyte-platelet aggregates (MPA) can regulate monocyte immune response. However the specific mechanism is still unclear. The formation of MPA in sepsis correlates with the release of a large number of inflammatory cytokines, and the CD40L-CD40 interaction is a prerequisite for MPA formation. CD40-dependent proinflammatory cytokine production is rely on ERK1/2 activation in monocytes. Based on this, the project proposed the hypothesis, when sepsis occurs, platelets and monocytes form MPA through CD40L-CD40 activate MAKP/ERK signaling pathway to promote the transcription of NF-kB to release inflammatory mediators in monocytes and mediate tissue and organs damage in the early stage of sepsis. This project aims to observe the formation of MPA by flow cytometry and its effect on monocyte inflammatory mediator secretion function. Using antibody block CD40L-CD40 interaction as well as siRNA in vitro to verify whether it is the key mechanism of platelet upregulation monocyte immune response. Also, this project aims to detect MAKP / ERK and p65 protein phosphorylation to explore the CD40L-CD40 downstream signaling pathways. This project will help deepen our understanding of platelet-mediated immune function and our understanding of the inflammatory-immune-mediated pathophysiological processes in sepsis, leading to new ideas and potential intervention targets for immunotherapy in early cytokine storm-mediated organ failure in sepsis.

脓毒症时血小板可通过与单核细胞形成聚集体（MPA）调控单核细胞免疫应答，但具体机制不清。MPA的形成与大量炎症介质释放相关；CD40L-CD40结合是MPA形成的必要条件；CD40依赖的炎症因子产生需要单核细胞中ERK1/2的活化。基于此，本项目提出假说：脓毒症时血小板与单核细胞形成MPA通过CD40L-CD40激活单核细胞MAKP/ERK信号通路促进NF-kB转录释放炎症介质，介导脓毒症早期组织损伤。本项目拟通过流式细胞术观察MPA的形成；抗体体外阻断及siRNA干扰验证CD40L-CD40是否是核心机制；检测MAKP/ERK和p65蛋白磷酸化程度，探究CD40L-CD40下游信号通路。本项目将有助于深化对血小板介导的免疫功能的认识，加深对脓毒症时炎症-免疫反应介导的病理生理过程的理解，为脓毒症免疫治疗提供新的思路和潜在干预靶点。

脓毒症时血小板可通过与单核细胞形成聚集体（MPA）调控单核细胞免疫应答，但具体机制不清。本项目提出假说：脓毒症时血小板与单核细胞形成MPA通过CD40L-CD40激活单核细胞MAKP/ERK信号通路促进NF-kB转录释放炎症介质，介导脓毒症早期组织损伤。本项目通过临床前瞻性观察性临床研究证实，脓毒症患者体内MPA水平明显升高，与外周血中炎症介质水平存在正相关；通过构建小鼠脓毒症模型后发现，血小板清除小鼠脓毒症造模后，外周血MPA减少的同时，小鼠病死率下降，炎症因子表达水平下调;利用抗体阻断CD40L与CD40的相互作用，以及利用siRNA干扰技术，PLPs或THP-1预封闭后将两种细胞共培养，检测上清中炎症介质TNF-a水平和IL-1b水平可以发现，阻断CD40L-CD40相互作用可以部分抑制PLPs上调的单核细胞炎症介质分泌作用；分选MPA后，检测MEK1/2、ERK1/2水平的变化，检测核蛋白中MSK1和p65蛋白水平和磷酸化的变化，无论是体外还是体内试验均证实，anti-CD40L mAb预处理后，NF-kB信号通路活性下降，提示MPA形成后通过CD40-CD40L相互作用依赖于MAKP/ERK信号通路激活发挥下游功能。本项目将有助于深化对血小板介导的免疫功能的认识，加深对脓毒症时炎症免疫反应介导的病理生理过程的理解，为脓毒症免疫治疗提供新的思路和潜在干预靶点