Shibata Masa-Aki, Shibata Eiko, Maemura Kentaro, Kondo Yoichi, Harada-Shiba Mariko
Department of Anatomy and Cell Biology, Division of Life Sciences, Osaka Medical College, 2-7, Daigaku-machi, Takatsuki, Osaka, 569-8686, Japan.
Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita, Osaka, 565-8565, Japan.
Med Mol Morphol. 2017 Sep;50(3):130-144. doi: 10.1007/s00795-017-0154-y. Epub 2017 Feb 28.
The establishment of consistent and reliable methods for the analysis of atherosclerosis molecular pathways and for testing the efficiency of new therapeutics is of utmost importance. Here, we fed ApoE-knockout (KO) mice with high-fat diet to for 16 weeks to induce atherosclerosis. Atherosclerotic lesions in mice were methodically investigated using pathologic analyses and molecular biology tools. These lesions were histopathologically classified into three categories: early, progressive, and combined lesions. Immunohistochemical analyses showed that both F4/80 (macrophage marker) and tenascin-C are expressed in these lesions. Real-time PCR analysis conducted using formalin-fixed paraffin-embedded tissues with atherosclerotic lesions demonstrated an increase in the levels of many inflammatory chemokines, including Cxcl16, while antibody arrays performed using frozen atherosclerotic tissue samples showed elevated TIMP-1 expression. Subsequent immunohistochemical analyses showed that the expression of CXCL16, TIMP-1, MMP-9, MMP-8, and LOX-1 is localized in the atherosclerotic lesions. We confirmed that the expression of these proteins is localized to atherosclerotic lesion, which suggests their roles in the development of the lesions in ApoE-KO mice. Therefore, this mouse model represents an appropriate tool for elucidating molecular mechanisms underlying the development of atherosclerosis, and a model for the evaluation of therapeutic efficiency of novel drugs.
建立用于分析动脉粥样硬化分子途径和测试新疗法疗效的一致且可靠的方法至关重要。在此，我们给载脂蛋白E基因敲除（KO）小鼠喂食高脂饮食16周以诱导动脉粥样硬化。使用病理分析和分子生物学工具对小鼠的动脉粥样硬化病变进行了系统研究。这些病变在组织病理学上分为三类：早期、进展期和复合型病变。免疫组织化学分析表明，F4/80（巨噬细胞标志物）和肌腱蛋白-C在这些病变中均有表达。使用含有动脉粥样硬化病变的福尔马林固定石蜡包埋组织进行的实时PCR分析显示，包括Cxcl16在内的许多炎性趋化因子水平升高，而使用冷冻动脉粥样硬化组织样本进行的抗体芯片分析显示TIMP-1表达升高。随后的免疫组织化学分析表明，CXCL16、TIMP-1、MMP-9、MMP-8和LOX-1的表达定位于动脉粥样硬化病变中。我们证实这些蛋白质的表达定位于动脉粥样硬化病变，这表明它们在载脂蛋白E基因敲除小鼠病变发展中的作用。因此，该小鼠模型是阐明动脉粥样硬化发展潜在分子机制的合适工具，也是评估新药治疗效果的模型。
