利用转基因兔模型研究基质金属蛋白酶－9在动脉硬化斑块破裂中的作用

The accumulation of macrophage-derived foam cells under the intima of large arteries is a hallmark of human and experimental animal atherosclerosis. Macrophages secrete a variety of proteinases that are thought to participate in vascular remodeling of the extracellular matrix (ECM) associated with all stages of atherosclerosis. Increased proteolysis of ECM molecules is probably involved in the initial mononuclear leukocyte emigration from the vascular lumen through the basement membrane into the subendothelial space, the migration and proliferation of medial smooth muscle cells through the elastic laminae in the intima, plaque destabilization, and rupture. But which one of matrix metalloproteinases (MMPs) is the key player in the pathogenesis of atherosclerotic plaque rupture has unknown. Our early results showed that MMP-9 is most important than other MMPs in unstable atherosclerotic lesions; thus we postulated that macrophage-derived MMP-9, in concert with other MMPs, may play a central role in the progression of atherosclerotic lesion and plaque rupture. However, the pathological roles of MMP-9 in lesions have not been defined. First ransgenic (Tg) rabbit overexpressing human MMP-9 will be generated in our laboratory. We can compare the susceptibility of MMP-9 transgenic (Tg) rabbits maintained at higher levels of hypercholesterolemia for longer periods to cholesterol-rich diet–induced atherosclerosis with that of non-Tg littermate rabbits. And we will generate transgenic (Tg) Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHL-MI) rabbits overexpressing human MMP-9 and compared their plasma lipids and aortic atherosclerosis with non-Tg WHHL-MI rabbits. It is also to be expected that Tg rabbits containe prominent macrophage infiltration associated with disruption of fibrotic cap with occasional formation of aneurysm-like or atherothrombotic lesions. This study not only shed fresh light on the functional roles of MMP-9 but also has implications for the notion that specific inhibition of MMP-9 activity may be a candidate therapeutic target for the treatment of atherosclerosis and its complications in the future.

巨噬细胞由来的泡沫细胞蓄积是动脉硬化斑块的主要特征。而由巨噬细胞分泌的基质金属蛋白酶对病变部血管壁重塑也起到重要作用。不单促进病变的增长，而且也增加动脉硬化斑块的不稳定性并可引起斑块破裂。哪种MMP是斑块破裂的关键现在还不明确。我们的研究显示，相对于其他巨噬细胞分泌的MMP，在不稳定性病变中MMP-9更为重要。因此推测巨噬细胞由来的MMP-9协调其他的MMPs在斑块破裂形成中起到核心作用，但其机制不详。我们将制作MMP-9的过表达转基因兔。对转基因组兔与对照组附加长期间高胆固醇饮食使其患高脂血症，并继续将其与家族性高脂血症心肌梗死兔模型（WHHL-MI兔）传代，创建MMP-9转基因WHHL-MI兔模型，系统分析各组动物的脂质代谢与病变特征。预期如得到类急性冠脉综合症病变结果或斑块破裂将验证我们的假说。此研究将为动脉硬化及其并发症的防治带来新思路，并建立与临床接近的新型易损斑块实验动物模型。

巨噬细胞由来的泡沫细胞蓄积是动脉硬化斑块的主要特征。由巨噬细胞分泌的基质金属蛋白酶(MMPs)对病变部血管壁重塑也起到重要作用，不单促进病变的增长，而且也增加动脉硬化斑块的不稳定性并可引起斑块破裂。我们的研究显示，相对于其他巨噬细胞分泌的MMPs，在不稳定性病变中MMP-9更为重要。为此，首先本项目开展了巨噬细胞特异表达的人MMP-9转基因兔的制作、分析与传代繁殖；利用MMP-9转基因兔开展动脉粥样硬化病变不同阶段造模实验与分析。结果表明MMP-9过度表达可加速Tg兔的动脉粥样硬化程度，尤其是中膜病变的恶化，提示巨噬细胞源性基质金属蛋白酶-9参与动脉粥样硬化的发生发展过程。并发现MMP-9转基因兔的主动脉和冠状动脉呈现高度进展的动脉硬化复合病变，且钙化明显增加，推测巨噬细胞源性MMP-9高表达介导了一个潜在的动脉硬化病变的破骨分化机制。其次开发了低密度脂蛋白受体（LDLr）、APOE等基因敲除家兔。正常饮食下LDLR基因敲除兔血浆胆固醇分析的结果表明，血浆中总胆固醇（TC）、甘油三脂（TG）以及低密度脂蛋白胆固醇（LDL-C）均有不同程度的提高，而高密度脂蛋白胆固醇（HDL-C）均有明显的下降。兔主动脉和冠状动脉均产生严重的动脉粥样硬化病变，严重程度与血脂高低成正相关，且部分伴有严重的黄色瘤。该系列家兔为今后深入研究家族性高胆固醇血症、动脉粥样硬化以及新一代降血脂药物的研发提供了合适的疾病动物模型，具有广泛的临床转化前景。此外围绕MMP-9与动脉粥样硬化斑块稳定性及相关疾病、中药单体干预动脉粥样硬化模型、脂质代谢相关microRNA等开展了系列研究，发现临床上MMP-9体内水平对斑块稳定性至关重要，是未来心脑血管疾病防治的较好干预靶点。本项目的相关研究为动脉硬化及其并发症的防治带来新思路，并建立了与临床接近的新型疾病实验动物模型。