ANIMAL MODELS OF HYPERTHYROIDISM

甲亢动物模型

• 批准号: 6571318
• 负责人: Sandra M McLachlan
• 金额: $ 35.96万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-15 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6571318
• 关键词: B lymphocyte; CHO cells; SDS polyacrylamide gel electrophoresis; T lymphocyte; autoantibody; biological signal transduction; cellular immunity; cytokine; disease /disorder model; enzyme linked immunosorbent assay; flow cytometry; gene targeting; genetically modified animals; hormone receptor; hyperthyroidism; laboratory mouse; polymerase chain reaction; radioimmunoassay; thyrotropin

DESCRIPTION (provided by applicant): Graves' disease is a common human autoimmune disorder caused by autoantibodies that stimulate the TSH receptor (TSHR). There are no spontaneous animal models of the disease. We will use two induced mouse models; "naked" TSHR-DNA vaccination, and the "Shimojo" approach (injecting TSHR-expressing fibroblasts). With these models, we will analyze the following issues in the immune response to the TSHR:- 1. Influence of micro-organisms or lack of self tolerance: We will study the titers and functional activity of TSHR antibodies induced by TSHR-DNA vaccination in combination with microbial products, as well as in TSHR-knockout mice that cannot acquire self tolerance to the TSHR. 2. Cytokine and cellular interactions: Wild type mice and B-cell knockout mice will be used to determine (a) which cytokines are produced by splenocytes challenged with TSHR-protein; (b) whether CD4+ or CD8+ T cells are involved in the response; (c) if B cells are required to induce memory T cells specific for the TSHR, 3. Epitopes recognized by TSHR-specific T cells: Synthetic TSHR peptides will be used to determine the epitopes recognized by T cells (a) cloned from TSHR-DNA vaccinated BALB/c mice; (b) TSHR knockout mice that lack tolerance to the TSHR; (c) mice transgenic for HLA that predispose (DR3) or protect against (DQ6) Graves' disease. Naturally processed TSHR peptides will also be studied, 4. Co-stimulatory signals: The role of co-stimulatory molecules will be explored (a) in vitro by using antibodies to block CD40/CD40-1igand and CD28/B7-1/2 interactions; (b) in vivo using mice with disrupted genes ("knockouts") for CD28, CD40, B7-1 or B7-2; (c) in vivo by injecting mice with anti-B7-1 (or control) together with TSHR -fibroblasts (that express B7-1). 5. Role of TSHR cleavage and shedding: We will test the hypothesis that TSHR cleavage followed by shedding of the heavily glycosylated A subunit plays a role in the immune response to the TSHR:- (a) using DNA encoding a non-cleaving or shedding TSHR; (b) examining binding of the heavily glycosylated TSHR A-subunit vs less glycosylated antigens (such as TPO) to the mannose receptor that captures glycosylated organisms for presentation to T cells.

描述（由申请人提供）：格雷夫斯病是一种常见的人类自身免疫性疾病，由刺激TSH受体（TSHR）的自身抗体引起。目前还没有该疾病的自发动物模型。我们将使用两种诱导小鼠模型； “裸”TSHR-DNA 疫苗接种，以及“Shimojo”方法（注射表达 TSHR 的成纤维细胞）。通过这些模型，我们将分析 TSHR 免疫反应中的以下问题：- 1.微生物的影响或缺乏自我耐受性：我们将研究TSHR-DNA疫苗接种与微生物产品联合诱导的TSHR抗体的滴度和功能活性，以及​​在无法获得TSHR自我耐受性的TSHR敲除小鼠中的滴度和功能活性。 2. 细胞因子和细胞相互作用：野生型小鼠和 B 细胞敲除小鼠将用于确定 (a) 哪些细胞因子是由 TSHR 蛋白攻击的脾细胞产生的； (b) CD4+或CD8+ T细胞是否参与反应； (c) 如果需要 B 细胞来诱导 TSHR 特异性的记忆 T 细胞， 3. TSHR 特异性 T 细胞识别的表位：将使用合成的 TSHR 肽来确定 T 细胞识别的表位 (a) 从接种 TSHR-DNA 的 BALB/c 小鼠中克隆； (b) TSHR 敲除小鼠对 TSHR 缺乏耐受性； (c) HLA 转基因小鼠，易患 (DR3) 或预防 (DQ6) 格雷夫斯病。天然加工的 TSHR 肽也将被研究， 4. 共刺激信号：将探索共刺激分子的作用（a）在体外使用抗体阻断CD40/CD40-1配体和CD28/B7-1/2相互作用； (b)在体内使用具有CD28、CD40、B7-1或B7-2基因被破坏（“敲除”）的小鼠； (c)体内注射抗B7-1（或对照）和TSHR-成纤维细胞（表达B7-1）的小鼠。 5. TSHR 裂解和脱落的作用：我们将检验以下假设：TSHR 裂解后重度糖基化的 A 亚基脱落在对 TSHR 的免疫反应中发挥作用：- (a) 使用编码非裂解或脱落 TSHR 的 DNA； (b) 检查高度糖基化的 TSHR A 亚基与较少糖基化的抗原（例如 TPO）与捕获糖基化生物体以呈递给 T 细胞的甘露糖受体的结合。