心肌激活型巨噬细胞防治心梗后心脏破裂的机制研究

Sudden cardiac death resulted from cardiac rupture associated with acute myocardial infarction (AMI-CR) remains no effective prevention and treatment. It is known that macrophages (M) is involved in the cardiac regeneration and repair. However, whether there is an interaction between M and cardiomyocyte (CM) remains unknown. We have previously demonstrated that c-kit is required for CM terminal differentiation (TD) and that adult transgenic C57BL/6J mice with CM-restricted overexpression of the dominant-negative Wv mutant (Tg) retain the ability of their CMs to proliferate; a response normally restricted to fetal and early neonatal life. In our pilot studies, Tg mouse hearts have been shown to resist AMI-CR. compared to WT controlnon-transgenic littermates, Tg mice displayed i) persistent CM replicative capacity in the adulthood, ii)increased left ventricular (LV) enlargement wall thickness and decreased end-diastolic volume due to increased CM cell number, and iii) at three months post-MI, WT hearts had undergone significant LV dilatation with thinning of the infarcted LV wall, whereas Tg LV chambers increased in length without LV wall thinning. This was associated with increased CM cell cycle entry, and infarct size was half that in WT hearts. The gene microarray and gene pedigree analysis showed that many M activation and polarization genes are up-regulated in Tg heart. We hypothesize that CM has a modulatory role in M and might be able to transform it into CM activated M (CAM), and CAM subsequently supports CM proliferation and improves cardiac wound repair. To test this hypothesis, we propose four specific aims：1) to investigate the interactions between the TD or non-TD cardiomyocyte with M and their roles in CR and cardiac repair after AMI; 2) by using the parabiosis model between (GFP ±TG mouse) and (WT mice ± AMI), to make tracks for the GFP+M origin; 3) to determine mRNA and miRNA expression profiles of myocardial activated M; 4) to illuminate the regulatory function of GDF15 in M1 to M2 transition signal pathway. The findings of this study will shed a new light on the mechanics of cardiomyocyte-activated macrophage in prevention of cardiac rupture post myocardial infarction and may also provide novel targets for the therapy.

巨噬细胞（M）参与急性心梗(AMI)后的再生修复，敲除M显著增加AMI后死亡率。此前，我们报道心肌细胞特异性封闭c-KIT功能的转基因（Tg）鼠的心肌细胞，于体内外均可增殖，为在体/离体心肌细胞与M间的互动研究提供了基础。预实验发现，Tg小鼠无心梗后心脏破裂。其心脏基因表达谱，表现出诸多影响M活性和转型的基因上调(如GDF15达19倍)，提示心肌细胞与M间互动可能使M变为心肌激活型M。故本项目研究以期：1）确定终极或未终极分化心肌细胞与M间的互动关系及其抗心脏破裂作用；2）应用GFP鼠±Tg鼠与野生鼠间的异种共生模型，确定心梗刺激下未终极分化心肌细胞释放的M激活因子，并作心肌激活型M起源的GFP荧光细胞标记追踪；3）研测心肌激活型M的mRNA和miRNA表达谱，以确定靶分子；4）确定GDF15对M1至M2转型信号通路调控机制。本研究将为心脏破裂的防治提供新的研发点以及靶细胞和靶分子。

急性心肌梗死（AMI）性心脏破裂的住院死亡率接近30％，其病情凶险，目前尚无有效的防治方法。本项目研究将在含未终极分化心肌细胞(CM)的Tg鼠作急性心梗模型，以期确定终极或未终极分化CM与巨噬细胞（M）间的互动关系及其抗AMI后心脏破裂的作用。应用GFP鼠±Tg鼠与野生鼠间的异体共生模型，确定AMI刺激下未终极分化CM释放的M间激活因子，并作心肌激活型M间起源的血液GFP细胞标记追踪研究。研测心肌激活型M间的mRNA和miRNA表达谱，以确定靶分子。确定GDF15对M1至M2转型信号通路调控机制。本项目研究发现，Tg鼠于早期抗心脏破裂的作用，可能部分归因于其前置性生理性CM肥大作用。进一步研究发现，Tg心脏的M的数量是WT小鼠心脏的两倍。在Tg心脏，其AMI后第三天的M主要为CD68—CD163+ M2亚型。我们发现1）Tg鼠在AMI后的存活曲线较WT鼠好；2）Tg鼠的心室壁在AMI后较WT的修复好；3）Tg心脏在AMI后的血管内皮细胞和成纤维细胞增生较WT多；4）Tg鼠的心脏功能较WT好。在敲除Tg小鼠心脏的M后，其对AMI后的心脏保护作用随之丧失；5）单核细胞不参与心脏常驻M的生成。在心脏，GM-CSF，CSF-1R，CD274, GDF-15和CXCL-10等5个基因上调。GDF-15和GM-CSF对M产生极化作用，增加M2极化的标记蛋白Arg-1的基因表达。故在AMI早期应用M2巨噬细胞，即可促进心脏损伤的修复，防治AMI后心脏的破裂。AMI后高血糖可导致患者预后不良，其机制不详。我们的研究发现外源性给予葡萄糖升高血糖1小时后，在正常鼠即可造成循环固有免疫细胞急性减少，而高糖所造成的细胞过氧化物累积而致细胞死亡是其原因。抗氧化剂谷胱甘肽(GSH)可完全阻止细胞的死亡。因此，对于AMI患者，及时给予GSH当可阻止糖毒性所造成的CM损害，有助患者的病情的康复。患者在给予葡萄糖输液时也应该同时加上GSH，以预防糖毒性所导致的细胞损伤。