血小板触发内皮细胞管腔面PECAM-1“扩散性诱陷”介导肺毛细血管漏的机制研究

Capillary leakage was the main reason of acute respiratory distress syndrome（ARDS）, which is short of the effective therapeutic strategies until now. Studies have demonstrated PECAM-1 was ‘captured’ at cellular con-junction to keep endothelial barrier by “diffusion trapping” of endothelial PECAM-1. However, PECAM-1 would redistribute to luminal surface under insult of inflammation, and platelet adhered to lung endothelium via PECAM-1-PECAM-1 interaction. As a result, endothelial junctions were broken to induce ARDS, as we found in our pilot study. Thus we hypothesized that platelet mediated pulmonary capillary leakage via triggering “diffusion trapping” in the pulmonary endothelial luminal PECAM-1. To test the hypothesis, PECAM-1+ platelets would be injected into mice to investigate the effect of PECAM-1+ platelet on pulmonary capillary leakage. Then PECAM-1 on pulmonary vascular endothelial cells would be silenced or over-expressed to determine whether PECAM-1 on endothelial cells is involved in capillary leakage via triggering “diffusion trapping”. At last, PECAM-1 signal pathways in both platelet and endothelial cells would be blocked to elucidate the mechanisms of PECAM-1 in pulmonary capillary leakage. Our research would be helpful to explore the effective intervention targets and strategies for ARDS.

肺血管内皮细胞连接损伤导致的毛细血管漏是ARDS的主要原因，尚无有效治疗措施。研究发现内皮细胞PECAM-1存在独特的“扩散性诱陷”效应，生理情况下均被“诱陷”分布于细胞连接处，而在炎症刺激下，PECAM-1可再分布至管腔面。申请者前期研究发现，急性肺损伤时血小板PECAM-1黏附于肺血管内皮细胞管腔面PECAM-1，导致内皮细胞连接断裂。据此提出：血小板触发血管内皮细胞管腔面PECAM-1“扩散性诱陷”效应，介导肺毛细血管漏的假说。本研究拟通过输注PECAM-1+血小板，观察活体肺组织，研究血小板是否参与肺毛细血管漏形成；通过沉默、过表达肺血管内皮细胞PECAM-1，研究PECAM-1+血小板通过管腔PECAM-1“扩散性诱陷”发挥作用；最后阻断管腔PECAM-1信号传导通路研究其诱发肺毛细血管漏的机制，为调控毛细血管漏及治疗ARDS提供新的理论依据。

脓毒症常伴随重要脏器屏障功能损伤，降低患者预后。在肺脏，血气屏障损伤引起的毛细血管漏，是诱发（Acute Respiratory Distress Syndrome，ARDS）的关键环节；在肠道，血肠屏障损伤可增加菌群转移，是加重脓毒血症、诱发多器官功能衰竭的重要因素。然而，屏障功能损伤的原因及其相关治疗措施仍有待进一步研究。血小板是维持屏障功能的关键血液成分，我们发现脓毒症中，血小板过度活化通过直接或间接作用损伤重要脏器屏障。在肺脏中，活化血小板可导致肺水渗出增加、小鼠生存率降低，该作用可被蛋白酶激活受体4（Protease-activated receptor 4，PAR4）拮抗剂抑制，但拮抗PAR1无效。拮抗PAR4可减少血小板在肺血管黏附数量。作为血小板黏附分子，拮抗P选择素对小鼠生存率及肺损伤无改善，但拮抗血小板内皮细胞黏附分子-1（Platelet endothelial cell adhesion molecule-1，PECAM-1）可减少血小板黏附，减少肺水渗出，提高小鼠生存率。且脓毒症时，PECAM-1参与血小板与肺血管相互作用，但P选择素并不与肺血管直接接触。综上，凝血酶通过PAR4激活血小板，活化血小板通过PECAM-1与肺血管相互作用，增加血气屏障通透性。同时，我们发现抑制血小板活化可增加肠道上皮细胞紧密连接蛋白表达，降低肠道屏障通透性，并降低CD40L+血小板比例和血液中CD40L水平。由于肠神经胶质细胞表达CD40，且体内95%的CD40L来自血小板，推测血小板通过CD40L-CD40信号通路调控肠道屏障功能。使用CD40-TRAF6抑制剂可模拟抗血小板药物效果，增加紧密连接蛋白表达，减少菌群移位，提高小鼠生存率。在体外，LPS和CD40L可刺激肠神经胶质细胞激活，其条件培养基可减少肠道上皮细胞系紧密连接蛋白表达，降低跨细胞电阻。沉默CD40可逆转上述作用，并减少肠神经胶质细胞释放GSNO，在体补充GSNO可改善肠道屏障功能，提高脓毒症小鼠生存率。综上，活化血小板通过CD40L-CD40-TRAF6信号减少肠神经胶质细胞GSNO释放，增加肠道屏障通透性，抑制血小板活化可能具有潜在治疗作用。本课题以脓毒症脏器屏障功能保护为目标，发现抑制血小板活化可改善肺脏、肠道屏障功能，为脓毒症治疗提供了新策略和理论依据。