The Role of TNF alpha and Apoptosis in Type 1 Diabetes

TNF α 和细胞凋亡在 1 型糖尿病中的作用

• 批准号: 6891889
• 负责人: LI-FEN LEE
• 金额: $ 13.15万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6891889
• 关键词: I kappa B beta; NOD mouse; T cell receptor; apoptosis; biological signal transduction; gene expression; gene induction /repression; genetic promoter element; genetically modified animals; insulin dependent diabetes mellitus; laboratory mouse; microarray technology; newborn animals; pancreatic islet function; pancreatic islets; tumor necrosis factor alpha; western blottings

DESCRIPTION (provided by applicant): The proposal will focus on the role of physiological beta cell apoptosis and the role of tumor necrosis factor alpha (TNFalpha) in the development of type 1 diabetes (T1DM). Type 1 diabetes is an autoimmune disease resulting from specific destruction of the beta-islet cells of the pancreas. Several reports have shown that apoptosis of beta-cells may play a crucial role at specific time points during disease progression. The proposed studies will test whether diabetes can be decreased or prevented by blocking apoptosis in the critical 17-21 day neonatal period. These experiments will explore the in vivo effects of quantitatively and temporally controlled expression of anti-apoptotic protein in beta-islet cells using a tetracycline-regulated gene expression (Tet-on) system with the rat insulin promoter RIP-7. Separate experiments will study the effect of neonatal TNF and anti-TNF therapy on the activation, or prevention of activation of islet and regional lymph node dendritic cells. Studies by the mentor, Dr. McDevitt, and others have shown that treatment in the neonatal period with TNFalpha, a potent apoptotic inducer in several model systems, leads to an earlier onset and increased incidence of T 1DM in NOD mice. Further, neonatal anti-TNF treatment completely prevents T1DM in NOD mice. The molecular mechanisms are not well understood. Paradoxically, chronic TNFalpha exposure in adult mice decreases T cell receptor signaling, while adult TNFalpha blockade appears to increase T cell effector function. The specific aims include: (1) Generation of tetracycline-regulated gene expression (Tet-on) system; this will use RIP7-rtTA, TRE-myc-CrmA and TRE-myc-Bclxl constructs; (2) Generation and characterization of RIP-7-rtTA, TRE-myc-CrmA, and TRE-myc-Bclxl transgenic NOD mice; (3) Characterization of neonatal and adult in vivo TNF and anti-TNF exposure in NOD mice; (4) Analysis of the effects of chronic in vivo TNFalpha and anti-TNFalpha exposure on the TNFalpha and T cell receptor signaling cascades in BDC 2.5 T cell receptor transgenic mice, by gene expression profiling on cDNA microarrays and by specific immunoblotting with anti-phosphotyrosine and anti-phosphoserine. These studies will aid in understanding the molecular mechanism ofapoptosis and the role of TNF and anti-TNF in the development of T1DM, as well as aid in understanding the relationship between the TNF and TCR signaling cascades.

描述（由申请人提供）： 该提案将重点关注生理性β细胞凋亡的作用以及肿瘤坏死因子α（TNFα）在1型糖尿病（T1DM）发展中的作用。 1 型糖尿病是一种自身免疫性疾病，由胰腺 β 胰岛细胞的特异性破坏引起。一些报告表明，β细胞的凋亡可能在疾病进展过程中的特定时间点发挥至关重要的作用。拟议的研究将测试是否可以通过在关键的 17-21 天新生儿期阻断细胞凋亡来减少或预防糖尿病。这些实验将探索使用四环素调节基因表达 (Tet-on) 系统和大鼠胰岛素启动子 RIP-7 在 β 胰岛细胞中定量和时间控制抗凋亡蛋白表达的体内效果。单独的实验将研究新生儿 TNF 和抗 TNF 疗法对胰岛和区域淋巴结树突状细胞激活或预防激活的影响。 导师 McDevitt 博士和其他人的研究表明，在新生儿期使用 TNFα（几种模型系统中的强效细胞凋亡诱导剂）进行治疗，可导致 NOD 小鼠 T 1DM 较早发病并增加发病率。此外，新生儿抗 TNF 治疗可完全预防 NOD 小鼠的 T1DM。其分子机制尚不清楚。矛盾的是，成年小鼠长期接触 TNFα 会降低 T 细胞受体信号传导，而成年小鼠 TNFα 阻断似乎会增加 T 细胞效应功能。 具体目标包括：（1）构建四环素调控基因表达（Tet-on）系统；这将使用 RIP7-rtTA、TRE-myc-CrmA 和 TRE-myc-Bclxl 构建体； (2) RIP-7-rtTA、TRE-myc-CrmA 和 TRE-myc-Bclxl 转基因 NOD 小鼠的产生和表征； (3) NOD 小鼠中新生儿和成人体内 TNF 和抗 TNF 暴露的表征； (4) 通过 cDNA 微阵列上的基因表达谱和抗磷酸酪氨酸特异性免疫印迹，分析长期体内 TNFα 和抗 TNFα 暴露对 BDC 2.5 T 细胞受体转基因小鼠中 TNFα 和 T 细胞受体信号级联的影响和抗磷酸丝氨酸。这些研究将有助于了解细胞凋亡的分子机制以及TNF和抗TNF在T1DM发展中的作用，并有助于了解TNF和TCR信号级联之间的关系。