The Role of TNF alpha and Apoptosis in Type 1 Diabetes

描述(由申请人提供)： 该提案将侧重于生理性β细胞凋亡和肿瘤坏死因子α(TNFpha)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-胰岛细胞特异性破坏引起。一些报告表明，在疾病进展的特定时间点，β细胞的凋亡可能起着至关重要的作用。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。这些实验将探索利用四环素调节基因表达系统(Tet-on)和大鼠胰岛素启动子RIP-7在体内定量和时间控制抗凋亡蛋白在β-胰岛细胞中的表达。单独的实验将研究新生儿肿瘤坏死因子和抗肿瘤坏死因子治疗对胰岛和区域淋巴结树突状细胞激活或防止激活的影响。 导师麦克德维特博士和其他人的研究表明，在新生儿期使用TNFpha治疗，在几个模型系统中都是一种有效的细胞凋亡诱导剂，导致NOD小鼠更早发病并增加T1 DM的发病率。此外，新生儿抗肿瘤坏死因子治疗完全预防NOD小鼠的T1 DM。其分子机制还不是很清楚。矛盾的是，成年小鼠长期暴露于TNFpha会降低T细胞受体信号，而成年TNFpha阻断似乎会增加T细胞效应器的功能。 具体目标包括：(1)建立四环素调节基因表达(Tet-On)系统，该系统将使用RIP7-RTTA、tre-myc-CrmA和tre-myc-Bclx1构建；(2)RIP-7-RTTA、tre-myc-CrmA和tre-myc-Bclx1转基因NOD小鼠的建立和鉴定；(3)新生和成年NOD小鼠体内肿瘤坏死因子和抗肿瘤坏死因子暴露的特征；(4)通过基因芯片表达谱和抗酪氨酸、丝氨酸特异性免疫印迹等方法，分析慢性体内暴露对BDC 2.5T细胞受体转基因小鼠肿瘤坏死因子α和T细胞受体信号转导通路的影响。这些研究将有助于了解细胞凋亡的分子机制以及肿瘤坏死因子和抗肿瘤坏死因子在T1 DM发生发展中的作用，并有助于了解肿瘤坏死因子和TCR信号转导通路之间的关系。

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.