小鼠肺移植早期再灌注损伤中NETs作用的可视化研究

项目名称： 小鼠肺移植早期再灌注损伤中NETs作用的可视化研究

项目编号： No.81470275

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 王兴安

作者单位： 同济大学

项目金额： 75万元

中文摘要： 补体主导的免疫攻击是再灌注损伤主因，但抑制补体的诸多尝试差强人意。我们假设，补体激活因供者肺泡巨噬细胞受损、中性粒细胞外陷阱(NETs)滞留而盛，随受者单核细胞分化为巨噬细胞、NETs被清除而衰。在B6=>B6肺移植，首先确定NETs滞留与持续补体激活的关联。A1-A3组供肺分别经历1h、24h、72h冷缺血，再灌注不同时间点获取标本。活体多光子荧光显微成像观察NETs附近巨噬细胞与中性粒细胞动态变化。其次，改变巨噬细胞吞噬功能或局部核酸酶水平，观察其对NETs和补体激活的影响。B1组取肺前诱导供者巨噬细胞凋亡，B2组受者再灌注后立即输注巨噬细胞。B3组关胸前胸内滴注DNase 1。最后用肺再移植模型模仿体外灌流装置，受损肺在第一受者调理24h，再植入第二受者后损伤有望减轻。本研究将明确肺巨噬细胞损伤-NETs滞留-补体持续亢进的关键机制，NETs有望成为肺再灌注损伤治疗靶。

中文关键词： 缺血再灌注损伤；肺移植；中性粒细胞外陷阱；多光子荧光显微技术；活体成像

英文摘要： Reperfusion injury (RI) is mainly attributed to complement-mediated immune attack, but various attempts to inhibit complement have so far been disappointing. We hypothesize that complement activation is thriving as Neutrophil Extracellular Traps (NETs) cannot be removed in time by impaired donor alveolar macrophages, and fading as more and more recipient -derived monocytes are developing into alveolar macrophages in graft. Firstly, the correlation between NETs overstocking and persistent complement activation is detected in B6=>B6 isogenic grafts with varying damage. In group A1, A2 and A3, The donor lungs suffer from 1h, 24h or 72h of cold ischemia (CI) respectively. Recipients will be sampled at 2h, 4h, 8h, 24h, 3d and 7d after reperfusion. The dynamics of macrophages and neutrophils near NETs will be measured by intraviatl multiphoton imaging,FACS and ELISA。Secondly, the consequence on NETs and complement activation will be observed by changing macrophage phagocytic function or local nuclease level. In group B1 (1h CI+24h reperfusion), apoptosis is induced in donor alveolar macrophages 4d and 1d before lung harvest. In group B2 (72h CI+24h reperfusion), 5×105/100ul alveolar macrophages will be adoptively transferred to recipients immediately after reperfusion.I n group B3 (72h CI+24h reperfusion), 50ug/50ul of DNase 1 will be instilled into chest cavity before closing. The behaviour of macrophages and neutrophils near NETs will be observed. Finally, the feasibility of targeting NETs will be tested in a model of lung retransplantation. RI in the second recipient is expected to be ameliorated greatly by preconditioning in the first recipient, a mimic of Ex vivo lung perfusion device. Our study will answer if impaired NETs degradation is a critical upstream event in RI, and if NETs is a potential target to prevent complement from being over activated.

英文关键词： Reperfusion injury;Lung transplantation;Neutrophil Extracellular Traps (NETs);Multiphoton Fluorescence Microscopy;intravital imaging