The Role of TNF alpha and Apoptosis in Type 1 Diabetes

描述（由申请人提供）： 该提案将重点关注生理性β细胞凋亡和肿瘤坏死因子α（TNF α）在1型糖尿病（T1 DM）发展中的作用。1型糖尿病是一种自身免疫性疾病，由胰腺的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期间的特定时间点起关键作用。拟议的研究将测试是否可以通过阻断关键的17-21天新生儿期的细胞凋亡来减少或预防糖尿病。这些实验将探索使用具有大鼠胰岛素启动子RIP-7的四环素调节的基因表达（Tet-on）系统在β-胰岛细胞中定量和时间控制的抗凋亡蛋白表达的体内作用。单独的实验将研究新生儿TNF和抗TNF治疗对胰岛和区域淋巴结树突细胞活化或预防活化的影响。 导师McDevitt博士和其他人的研究表明，在新生儿期用TNF α（几种模型系统中的强效凋亡诱导剂）治疗可导致NOD小鼠中T1 DM的早期发作和发病率增加。此外，新生儿抗TNF治疗完全预防NOD小鼠中的T1 DM。其分子机制还不清楚。有趣的是，成年小鼠的慢性TNF α暴露降低了T细胞受体信号传导，而成年TNF α阻断似乎增加了T细胞效应功能。 具体目标包括：（1）四环素调节的基因表达（Tet-on）系统的产生;这将使用RIP 7-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小鼠中新生和成年体内TNF和抗TNF暴露的表征;（4）通过cDNA微阵列上的基因表达谱和通过用抗磷酸酪氨酸和抗磷酸丝氨酸的特异性免疫印迹，分析长期体内TNF α和抗TNF α暴露对BDC 2.5 T细胞受体转基因小鼠中TNF α和T细胞受体信号传导级联的影响。这些研究将有助于理解三元共聚物的分子机制以及TNF和抗TNF在T1 DM发展中的作用，并有助于理解TNF和TCR信号级联之间的关系。

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.