巨噬细胞胱天蛋白酶募集域蛋白9(CARD9)调控心梗后损伤修复的机制研究

With high mortality rate, myocardial infarction (MI) is a severe type of coronary heart diseases. Infiltration of macrophages following acute MI is found to be a prerequisite of infarct healing and left ventricular remodeling. An appropriate healing process and formation of a scar with tensile strength can prevent infarct expansion and adverse ventricular dilation. Furthermore, Various subsets of macrophages are reported to play different roles in healing process of MI, and NF-κB signaling is thought to be a key piont in mediating the M1/M2 differentiation and functions of macrophages. Caspase-associated recruitment domain 9 (CARD9) commonly expressed in macrophages elicits innate immunity via activation of nuclear factor (NF)-κB signaling pathway. Nevertheless, it is lack of direct evidence concerning that CARD9 modulates macrophages differentiation and infarct healing of MI. Our preliminary results showed that knock-out of CARD9 promoted MI healing, with enhanced myofibroblast infiltration and collagen deposition, protecting from deterioration of cardiac function. Consequently, we hypothesize that CARD9 deletion promotes infiltrated macrophages swithing to M2 subset, which leads to enhancement of angiogenesis and myofibroblasts activation mediated healing following MI, through secreting high levels of anti-inflammatory cytokines and growth factors, such as transforming growth factor-β and VEGF, avoiding occurance of heart failure. In this study, we plan to validate our proposal in vivo through using of the established MI animal model and CARD9 gene knock-out mice. Moreover, co-culture of macrophages and fibroblasts and flow cytometry analysis of inflammaroty cells will be applied to investigate the mechanism underlying our preliminary findings. Within this study, we anticipate to explore a novel theraputic target of myocardial ischemic diseases.

心肌梗死是冠心病极危重的表现类型，死亡率高。急性心肌梗死后，巨噬细胞浸润介导修复与左心室重构，促进心梗后修复可有效地改善预后。不同亚型巨噬细胞在心梗修复中具有不同作用，而NF-κB信号途径参与了巨噬细胞的M1/M2亚型分化和功能调节。表达于巨噬细胞的胱天蛋白酶募集域蛋白9 (CARD9)可以通过调控NF-κB途径介导天然免疫，但CARD9在巨噬细胞分化和心梗修复中的作用及机制尚无报道。本课题组预实验提示CARD9敲除促进了心梗后修复，增加了梗死区胶原的沉积，改善了心功能。据此我们推测CARD9缺失可以通过抑制NF-κB激活，促进心梗后浸润的巨噬细胞向M2型分化，促进血管新生并激活肌成纤维细胞，推动梗死修复，阻止心衰发生。本课题拟用CARD9敲除小鼠和心梗模型在整体水平进行研究，并通过巨噬细胞与成纤维细胞共培养、流式细胞等手段进行机制探讨，以期为心脏缺血性疾病的治疗提供新思路。

心肌梗死是由于冠状动脉急性狭窄或闭塞所产生的心肌严重缺血和坏死，是冠心病极具危重的表现类型。心肌梗死起病急，发病凶险，死亡率高，预后差。心脏细胞缺血缺氧导致细胞溶胀死亡，细胞内容释放到周围的组织中，导致炎症细胞浸润，激活天然免疫过程。巨噬细胞作为一种重要的炎症细胞，其浸润从心梗后3天内到数周都会存在，巨噬细胞功能在心梗后损伤和修复过程中发挥关键作用。. 胱天蛋白酶募集域蛋白9 (CARD9)是一种介导巨噬细胞和树突状细胞参与天然免疫反应的信号蛋白。在本课题中我们发现其在心梗后损伤与修复中发挥重要作用。我们利用结扎小鼠左冠状动脉建立了小鼠急性心肌梗死模型，发现CARD家族中CARD9和CARD11表达显著上调。CARD9蛋白水平从心梗后1天开始升高，到7天开始消退；免疫荧光和流式细胞分析鉴定其定位于巨噬细胞。利用CARD9基因敲除小鼠，我们发现CARD9敲除可以明显改善心梗28天小鼠心功能，减少梗死面积。免疫组化染色和细胞因子检测，发现CARD9敲除不能促进交界区血管新生；但是减少了巨噬细胞浸润，降低了血浆和心脏组织炎症因子水平，并且抑制了细胞基质蛋白酶MMP-9的表达。为进一步研究其机制，体外利用坏死细胞刺激野生型和CARD9敲除巨噬细胞，发现敲除CARD9可以抑制炎症因子表达，这一作用是通过抑制NF-κB活化实现的。因此进一步减轻了心梗后3天梗死交界区心肌细胞的进一步凋亡。. 本课题揭示了巨噬细胞CARD9通过调控坏死细胞对NF-κB的活化，进而促进其分泌炎症因子的功能，促进心梗后的损伤，为缺血性心脏疾病的治疗提供新的靶点。