MINORITY PREDOCTORAL FELLOWSHIP PROGRAM

少数族裔博士前奖学金计划

• 批准号: 7230716
• 负责人: LUIS A ZUNIGA
• 金额: $ 4.1万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7230716
• 关键词: Adipose tissue; Anti-Inflammatory Agents; Anti-inflammatory; Antigen Presentation; Antigens; Aorta; Apolipoprotein E; Arginine; Arterial Fatty Streak; Atherosclerosis; Binding; Biological Assay; Blood; Body fat; Bone Marrow; Cells; Chemotactic Factors; Coagulation Process; Data; Development; Diet; Disease; Dual-Energy X-Ray Absorptiometry; Eating; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Fatty acid glycerol esters; Fellowship Program; Flow Cytometry; G-Protein-Coupled Receptors; Gene Expression; Goals; Harvest; Human; Immune response; Immunohistochemistry; Immunosuppressive Agents; In Vitro; Inflammation; Inflammatory; Label; Leukocyte Trafficking; Ligands; Lipids; Magnetic Resonance Imaging; Maintenance; Metabolism; Minority; Modeling; Mus; Nitric Oxide; Numbers; Obesity; Orthologous Gene; Peritoneal Macrophages; Personal Satisfaction; Phagocytosis; Plasma; Play; Population; Production; Public Health; Recruitment Activity; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Serine Protease; Site; Staining method; Stains; Sterility; Stimulus; Sudan; T-Cell Proliferation; T-Lymphocyte; Testing; Time; Tissues; Weight; arginase; chemokine; cytokine; fMet-Leu-Phe receptor; feeding; glucose tolerance; human CMKLR1 protein; in vivo; in vivo Model; insulin tolerance; intraperitoneal; macrophage; migration; mouse model; novel; oil red O; pre-doctoral; prevent; receptor expression; response; selective expression; therapeutic target; trafficking

DESCRIPTION (provided by applicant): The goal of this project is to characterize the mouse ortholog of Chemokine-Like Receptor 1 (CMKLR1) on macrophages and determine its role in obesity and atherosclerosis. Aim 1 proposes to characterize the expression of CMKLR1 on developing and tissue resident macrophages. Bone marrow derived macrophages (BMDM) will be assayed for receptor expression over time. Various tissue resident macrophages (TRM) will also be assayed for receptor expression. BMDMs and TRMs will be stimulated with pro-inflammatory and anti-inflammatory factors and assayed for CMKLR1 expression in response to said factors (flow cytometry, immunohistochemistry [IHC]). Aim 2 proposes to characterize the effect of functional CMKLR1 on basic macrophage effector functions either in the presence or absence of CMKLR1 ligand (chemerin). CMKLR1 KO or WT TRMs will be treated with or without chemerin and assayed for cell spreading, cytokine expression (ELISA or intracellular staining), nitric oxide production (Greiss assay), phagocytosis (FITC-labeled E. Coli), arginase activity (L-arginine metabolism), T cell antigen presentation ability (antigen T cell proliferation assay), and T cell costimulation ability (costimulation T cell proliferation assay) in response to various activating stimuli. Aim 3 will focus on characterizing the role of CMKLR1 in mouse models of obesity and athersclerosis. For obesity, CMKLR1 WT and KO mice will be fed a high fat diet and their weights/food intake recorded over time. At various time points, whole body fat content will be assayed (dual-energy-X-ray-absorptiometry or quantitiative MRI). Mice will be assayed for glucose tolerance (intraperitoneal glucose tolerance assay), insulin tolerance (intraperitoneal insulin tolerance assay), and plasma lipid (Reflotron test strips). Adipose tissue will be assayed for macrophage content (flow cytometry, IHC), proinflammatory gene expression (quantitative RT-PCR, tissue macrophage intracellular cytokine expression), and chemerin expression (IHC). For atherosclerosis, CMKLR1 WT and KO mice deficient for ApoE will be fed a normal diet. Periodically, mice will be sacrificed and their aortas harvested along with blood. Plasma levels of lipids with be assayed and aortic atheromatous plaques quantitated (Oil-Red-O or Sudan V staining). Aortic macrophage content will be analyzed (IHC) and pro- inflammatory gene expression (qRT-PCR) will be assessed. RELEVANCE: The proposed research is relevant to public health as macrophages play a large role in the development and maintenance of inflammatory diseases. The proposed research will endeavor to further elucidate the mechanisms by which macrophages infiltrate inflamed tissue, thus offering a potential therapeutic target.

描述(由申请人提供)：本项目的目标是鉴定巨噬细胞上趋化素样受体1(CMKLR1)的小鼠同源基因，并确定其在肥胖和动脉粥样硬化中的作用。目的1研究CMKLR1在发育中巨噬细胞和组织内滞留巨噬细胞上的表达。随着时间的推移，将检测骨髓来源的巨噬细胞(BMDM)的受体表达。各种组织驻留巨噬细胞(TRM)的受体表达也将被检测。用促炎和抗炎因子刺激BMDM和TRMS，并检测CMKLR1在上述因素作用下的表达(流式细胞仪、免疫组织化学[IHC])。目的2研究在CMKLR1配体(趋化蛋白)存在与否的情况下，功能性CMKLR1对巨噬细胞基本效应功能的影响。CMKLR1KO或WT TRMS将被用或不用趋化蛋白处理，并检测细胞扩散、细胞因子表达(ELISA或细胞内染色)、一氧化氮产生(Greiss法)、吞噬功能(FITC标记的大肠杆菌)、精氨酸酶活性(L-精氨酸代谢)、T细胞抗原提呈能力(抗原T细胞增殖实验)和T细胞共刺激能力(共刺激T细胞增殖实验)对各种激活刺激的反应。目的3将重点研究CMKLR1在肥胖和动脉粥样硬化小鼠模型中的作用。对于肥胖，CMKLR1 WT和KO小鼠将被喂以高脂肪饮食，并随着时间的推移记录它们的体重/食物摄入量。在不同的时间点，测定全身脂肪含量(双能X线吸收法或定量磁共振成像)。将对小鼠进行葡萄糖耐量(腹膜糖耐量试验)、胰岛素耐量(腹膜胰岛素耐量试验)和血脂(Reflutron试纸)的检测。脂肪组织将检测巨噬细胞含量(流式细胞仪，IHC)、促炎基因表达(定量RT-PCR、组织巨噬细胞细胞内细胞因子表达)和趋化蛋白表达(IHC)。对于动脉粥样硬化，ApoE缺乏的CMKLR1WT和KO小鼠将以正常饮食喂养。每隔一段时间，老鼠就会被处死，同时采集它们的动脉和血液。测定血脂水平和动脉粥样硬化斑块数量(油红O或苏丹V染色)。将分析主动脉巨噬细胞含量(IHC)和促炎基因表达(qRT-PCR)。相关性：拟议的研究与公共健康相关，因为巨噬细胞在炎症性疾病的发展和维持中发挥着重要作用。这项拟议的研究将努力进一步阐明巨噬细胞渗透炎症组织的机制，从而提供潜在的治疗靶点。