外源性CO通过GC/cGMP/PKG信号通路调控脓毒症时EC/SMC互调作用的机制研究

Microenvironment and two-way adjustment disorder between endothelial cell (EC) and vascular smooth muscle cells （SMC）during sepsis are important factors leading to dysfunction of blood vessel、inadequate tissue perfusionand Organ dysfunction, the detail mechanisms, however, are not completely elucidate. This project employs CLP mice and LPS-stimulated cells as sepsis models to examine the dynamic state of EC/SMC interaction under the intervention of the exogenous CO liberated by CO-releasing molecules. The important factors will be investigated in the molecular and cellular levels through applying updated molecular biological techniques including gene analysis, mass spectrometric analysis, measurement of protein expressions, which investigate the structural and functional changes of EC/SMC, microenvironment and two-way adjustment disorder between endothelial cell and vascular smooth muscle cells ,and essential regulator of EC/SMC such as ion channels, the dynamic changes of Notch signal transduction during sepsis. Furthermore, this project also focuses on the important influences of the shear stresses induced by instabal circulating fluidin sepsis. Therefor, we are in attempt to elucidate the correlation of microenvironment and two-way adjustment between endothelial cell and vascular smooth muscle cells, to determine whether sGC/cGMP pathway and Notch signal transduction will be the novel targets of exogenous CO, in order to elucidate molecular mechnisms (even partly) responsible for the regulatory effects of CO on abnormal microenvironment and two-way adjustment of EC/SMC and signal transduction during sepsis and to provide theoretical basis and new idea for the research and treatment of sepsis.

脓毒症时血管内皮细胞（EC）和平滑肌细胞（SMC）微环境失稳、互调过程紊乱是导致血管功能障碍和器官功能不全的重要因素，但详细机制尚未阐明。本研究采用CLP脓毒症动物模型和EC/SMC细胞单（共）培养模型，以外源性一氧化碳（CORM）进行干预，运用基因分析、质谱分析、蛋白质水平测定和器官功能评价等手段，在分子、细胞和整体等不同层面上，研究脓毒症时EC、SMC重要结构和功能、缝隙连接蛋白Cx、微环境失稳态变化，以及EC和SMC间重要的互调因素、信号通道GC/cGMP/PKG的动态变化，并首次利用原子力显微技术和正交相位显微细胞亚表面3D特征识别技术，精确观察脓毒症时SMC收缩表型，旨在探明脓毒症时EC/SMC微环境和双向调节功能之间的相关关系，探明外源性CO通过GC/cGMP/PKG途径调节脓毒症时异常的EC/SMC微环境和相互作用的（部分）分子学基础，为发现脓毒症干预新靶位提供新的思路。

脓毒症时血管内皮细胞（EC）和平滑肌细胞（SMC）、中性粒细胞微环境失稳、互调过程紊乱是导致血管功能障碍和器官功能不全的重要因素，但详细机制尚未阐明。本研究采用相关细胞模型，运用基因分析、质谱分析、蛋白质水平测定和器官功能评价等手段，在分子、细胞和整体等不同层面上，研究脓毒症时EC、SMC重要结构和功能的变，结果表明：1）LPS刺激可促进中性粒细胞穿越内皮细胞层, 内皮和中性粒细胞的直接接触以及内皮细胞的凋亡是中性粒细胞跨内皮迁移的关键。2） P2X1抑制剂可以明显降低中性粒细胞穿越内皮细胞。3） 中性粒细胞通过P2X1受体调节LPS刺激的内皮细胞的凋亡。4） P2X1调节的LPS刺激的中性粒细胞穿内皮，上清释放物质或产物并不发挥主要作用。P2X1为嘌呤能受体其受体本身以及与ATP的关系，对中性粒细胞和内皮的释放有很大的影响。5） P2X1可以调节LPS诱导的中性粒细胞粘附内皮。本实验提出了一个新的可能性观点，在较高浓度的LPS（10μg/mL）刺激下，中性粒细胞能够通过其与内皮细胞粘附，影响内皮细胞的凋亡，这在很大程度上使中性粒细胞从血管内迁移至血管外的能力及数量均明显增加，也许这就是脓毒症时，过量的中性粒细胞浸润正常组织，造成多器官功能障碍的机制之一。本研究揭示了P2X1受体在脓毒症时对内皮和中性粒细胞相互作用的影响，P2X1通过ICAM-1调节内皮细胞凋亡，部分抑制LPS增强的中性粒细胞跨内皮迁移作用。本课题初步阐明了脓毒症时内皮细胞重要的功能变化和中性粒细胞的特殊干预作用，为临床脓毒症治疗提供了新思路。