MINORITY PREDOCTORAL FELLOWSHIP PROGRAM

少数族裔博士前奖学金计划

• 批准号: 7646266
• 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/7646266
• 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）：该项目的目标是表征巨噬细胞上趋化因子样受体 1 (CMKLR1) 的小鼠直系同源物，并确定其在肥胖和动脉粥样硬化中的作用。目标 1 提出表征 CMKLR1 在发育中和组织驻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。还将检测各种组织驻留巨噬细胞 (TRM) 的受体表达。将用促炎和抗炎因子刺激 BMDM 和 TRM，并分析响应所述因子的 CMKLR1 表达（流式细胞术、免疫组织化学 [IHC]）。目标 2 旨在描述在存在或不存在 CMKLR1 配体（凯莫瑞）的情况下，功能性 CMKLR1 对基本巨噬细胞效应器功能的影响。 CMKLR1 KO 或 WT TRM 将用或不用凯莫瑞处理，并测定细胞扩散、细胞因子表达（ELISA 或细胞内染色）、一氧化氮产生（Greiss 测定）、吞噬作用（FITC 标记的大肠杆菌）、精氨酸酶活性（L-精氨酸代谢）、T细胞抗原呈递能力（抗原T细胞增殖测定）和T细胞共刺激能力（共刺激T细胞增殖测定）响应各种激活刺激。目标 3 将重点描述 CMKLR1 在肥胖和动脉粥样硬化小鼠模型中的作用。对于肥胖症，CMKLR1 WT 和 KO 小鼠将被喂食高脂肪饮食，并随时间记录它们的体重/食物摄入量。在不同的时间点，将测定全身脂肪含量（双能 X 射线吸收测定法或定量 MRI）。将测定小鼠的葡萄糖耐量（腹膜内葡萄糖耐量测定）、胰岛素耐量（腹膜内胰岛素耐量测定）和血浆脂质（Reflotron 测试条）。将检测脂肪组织的巨噬细胞含量（流式细胞术，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）和凯莫瑞表达（IHC）。对于动脉粥样硬化，缺乏 ApoE 的 CMKLR1 WT 和 KO 小鼠将以正常饮食喂养。定期处死小鼠并采集其主动脉和血液。测定血浆脂质水平并对主动脉粥样斑块进行定量（油红-O 或苏丹 V 染色）。将分析主动脉巨噬细胞含量（IHC）并评估促炎基因表达（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。拟议的研究将努力进一步阐明巨噬细胞浸润发炎组织的机制，从而提供潜在的治疗靶点。