HLA-A2抗体通过Src-钙信号通路诱发TRALI的作用及相关机制研究

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related death. Studies have shown that anti-HLA-A2 is closely related to TRALI occurrence, but the mechanism is unclear. Our previous study have showed that anti-HLA-A2 can raise polymorphonuclear neutrophils (PMN) adhesion properties, inflammatory cytokine secretion and intracellular calcium signaling. It is also reported that activation of PMN immune function are dependent on Src phosphorylation, suggesting that anti-HLA-A2 may activate PMN to induce TRALI through Src-Calcium signal pathway. Based on previous study, we plan to compare the secretion of adhesion characteristics and inflammatory cytokines of PMN with different treatment by combining inhibitors of key signaling molecules (Src or intracellular Ca2+) with flow chamber and real-time fluorescence technique, and also intend to compare calcium signaling and ROS of PMN with different treatment, and the apoptosis rate of pulmonary vascular endothelial cells by fluorescent labeling, cell culture and flow cytometry to clarify the pathogenesis that anti-HLA-A2 induced TRALI through upregulating PMN adhesion properties and inflammatory cytokine secretion and enhancing calcium response signal and ROS production by intracellular Src-Calcium signaling pathway to mediate pulmonary vascular endothelial cell injury, which to lay the theoretical foundation for the possible applications of the prevention of TRALI by targeting to anti-HLA-A2 and in the early warning and monitoring of TRALI.

输血相关急性肺损伤(TRALI)是导致输血相关性死亡的首要原因。有研究显示HLA-A2抗体（A2抗体）与TRALI发生密切相关，但机制不明。我们前期研究发现A2抗体能上调嗜中性粒细胞（PMN）粘附特性和炎性因子分泌及胞内钙信号，而现有研究已证实其有赖于PMN胞内Src磷酸化，提示A2抗体可能通过Src-钙信号通路诱发TRALI。本项目在前期基础上借助Src、Ca2+关键信号分子抑制剂，结合流动小室、荧光实时记录处理手段探讨不同条件下PMN粘附特性和炎性因子分泌的变化；采用荧光标记、细胞培养和流式细胞技术探寻不同条件下PMN钙信号、ROS与肺血管内皮细胞凋亡率的变化规律，以阐明A2抗体通过上调PMN粘附特性和炎性因子分泌及Src-钙信号通路增强钙响应信号而促进ROS产生，介导肺血管内皮细胞损伤诱发TRALI的发病机制，为以A2抗体为靶点防治及发病风险预警和病情监测应用奠定实验基础和理论依据。

输血相关急性肺损伤（TRALI）是导致输血相关性死亡的首要原因。研究显示HLA-A2抗体与TRALI发生密切相关，我们前期研究发现HLA-A2抗体能促进中性粒细胞（PMN）在肺组织粘附聚集并分泌炎性因子，但其分子机制并不清楚。.本研究在前期工作基础上，通过建立LPS联合HLA-A2抗体阳性血浆诱发TRALI的二次打击大鼠模型，证实了PMN在TRALI发生发展中发挥重要作用。细胞与动物实验研究显示，HLA-A2抗体可通过磷酸化Src诱导PMN胞内NF-κB转录激活并提高NLRP3炎性小体表达，促进炎性因子分泌；同时，实时荧光结合流动小室技术发现HLA-A2抗体激活Src-钙信号通路增强钙响应，促进PMN细胞骨架重排，上调PSGL-1、LFA-1表达和释放活性氧，诱导肺血管上皮细胞凋亡和通透性增加诱发TRALI。而Src抑制剂明显抑制HLA-A2抗体诱导的PMN粘附特性和炎性因子释放，降低上皮细胞凋亡和肺血管通透性，抑制TRALI的发生发展。.上述研究结果证实，PMN在TRALI发生发展中具有重要作用，而有研究表明，PMN活化、凋亡后形成NET，其是否参与了TRALI的发生发展及其机制尚不清楚，本研究进一步探讨了NET在TRALI发生发展中的可能机制。通过分析TRALI患者及大鼠模型血样，发现miR-144在TRALI患者、大鼠模型中高表达。动物实验表明，过表达miR-144可加剧TRALI的严重程度，且这一过程可能依赖于NET的形成。生物信息学分析并通过双荧光素酶报告基因及增益和功能丧失实验验证，miR-144可以通过靶向抑制KLF2表达诱导NET形成。通过构建了KLF2及其相关基因的PPI网络，Western blot验证相关基因，发现TRALI小鼠PMN中p65磷酸化和CXCR1蛋白水平显著升高，证实了TRALI模型中NF-κB信号通路被激活。据此，我们认为miR-144通过抑制KLF2的表达激活NF-κB信号通路，促进NET形成诱发TRALI，而NF-κB抑制剂可明显抑制miR-144诱发TRALI的发生发展。.本研究深入阐明了PMN中Src和NF-κB分子在TRALI发生发展中的分子机制，发现了抑制Src磷酸化可有效阻断HLA-A2抗体诱发的TRALI，以及抑制NF-κB可有效阻断miR-144诱发的TRALI，为TRALI的临床治疗提供了多角度的实验依据。