基于冠状动脉粥样硬化的小鼠冠心病模型构建及其人源化方法

Clinically, coronary atherosclerosis is the major pathogenesis of coronary heart disease. Herein, the formation of atherosclerotic plaque requires the activation of auto-immune T cells, which recognize the low-density lipoprotein as alloantigen. In basic research, however, the animal model of coronary heart disease is established through ligating the coronary artery in mice and other model animals and, in the other hand, no coronary atherosclerosis and cardiac dysfunction are detected in the hyperlipidemia mice. Therefore, a mouse model of coronary atherosclerotic heart disease that matches the clinical symptom is urgently needed. In our previous study, severe coronary atherosclerosis and heart failure were detected in high-fat diet-fed ApoE-/- mice receiving heterotopic cardiac transplantation. Based on this discovery, we propose a series of simplified transplantations into ApoE-/- mice with hyperlipidemia induction (treated with high-fat diet or PCSK9 adeno-associated virus), including myocardial tissue transplantation and cardiac cell injection intraperitoneally, and tail-vein injection of leukocytes from these cardiac-transplantation recipients. The best strategy and the corresponding standard operating procedures will be determined based on the evaluation of coronary atherosclerosis and cardiac function and mice survival curve. Also, we propose to establish a humanized atherosclerosis system in ApoE-/- mice with immunodeficient background (ApoE-/-NCG mice). The carrier mouse will receive a graft of human coronary branch into abdominal aorta and the tail-vein injection of peripheral blood mononuclear cells from the same donor, followed by hyperlipidemia induction. Hereby, the high blood lipid and the human mononuclear cells would give rise to atherosclerosis in the implanted human artery, which might reproduce the clinical process of coronary atherosclerosis. Furthermore, the role of T cells and their regulatory cytokines in coronary atherosclerotic heart disease will be investigated in the two types of models. The accomplishment of the current project will make up the lack of suitable animal models in the study of coronary heart disease and, more importantly, support the development of new diagnostic and therapeutic strategies for patients suffering from the disease.

临床上，冠状动脉（冠脉）粥样硬化是冠心病的主要病理基础，斑块的形成与发展依赖于识别低密度脂蛋白的自体免疫性T细胞在冠脉内的激活。但是，目前在小鼠等模式动物中冠心病模型通过结扎冠脉建立，高脂血症小鼠不发生冠脉粥样硬化和心功能障碍，因此都不能准确模拟临床。我们发现，器官移植会引发自体免疫性T细胞在小鼠冠脉内激活，导致冠脉粥样硬化的形成与发展。本课题将以高脂血症小鼠为受者，采用同种异体心肌组织、心脏细胞移植和受者外周血单个核细胞移植等方法探索最佳建模方案，建立标准化、易操作的基于冠脉粥样硬化的小鼠冠心病模型。我们还将以高脂血症免疫缺陷小鼠为载体，植入人源冠脉和同一供者外周血单个核细胞，重建匹配临床的人源化动脉粥样硬化系统。在这两类模型基础上，我们将研究T细胞及其调节因子在冠脉粥样硬化和冠心病中的作用。本课题的开展将弥补冠心病致病机理研究的动物模型缺陷，更将有力支撑冠心病诊疗新策略的精准研发。

随着人民生活水平的提高和平均寿命的延长，冠心病成为危害人们健康的头号杀手。目前常用的以手术结扎为主要手段的急性心肌梗死模型难以模拟以动脉粥样硬化为病理基础的冠心病。普通动脉粥样硬化小鼠模型粥样斑块较少发生再冠状动脉中，我们尝试通过异体移植的方法诱发小鼠的免疫反应提高小鼠动脉粥样硬化的严重程度以引发小鼠产生冠心病表现。我们考察了心脏细胞、胸骨、腿骨、骨髓细胞、脾脏细胞五种不同的异体移植方法，选定了骨髓移植为主要造模方法，并从移植次数、饲料是否含胆酸这两个方面进行改进，尝试构建有效且稳定的小鼠冠心病模型。实验发现ApoE-/-小鼠MHC完全错配的Balb/C小鼠骨髓细胞通过尾静脉注射方法移植入ApoE-/-小鼠体内，两周进行一次移植并以含胆酸的高脂饲料喂养可引起小鼠动脉粥样硬化情况的整体加重。以往的研究结合我们的多次尝试表明，小鼠独特的心脏冠状动脉结构（小鼠由左冠状动脉、右冠状动脉、室间隔冠状动脉三支营养心脏且侧枝循环丰富，而人类心脏仅有左冠状动脉和右冠状动脉）和极高的心跳频率，导致慢性冠心病模型在较短的时间内建立存在较大的挑战性。但骨髓移植引起的动脉粥样硬化严重程度上升可诱发小鼠出现冠状动脉中粥样斑块、心肌纤维化、心脏功能异常等冠心病表现。异种移植物诱导激活的T细胞不仅可以促进主动脉上粥样硬化斑块的形成，同时也可以促进冠状动脉中粥样硬化斑块的形成，并且是通过IL-18介导促进冠心病的发生发展。

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