Lysate Arrays for Studying the Diabetes Proteome

描述（由申请人提供）：拟议的研究旨在分析1型糖尿病（T1D）小鼠模型中转录组和蛋白质组的一个重要子集，特别是随着时间的推移表征胰腺淋巴结（PLN）细胞、胰岛β细胞和外周血细胞的转录组和蛋白质组。然后，我们将使用系统生物学方法通过抗cd3单克隆抗体治疗小鼠来干扰系统，该抗体已被证明可以逆转新糖尿病小鼠的高血糖，目前正在人体临床试验中。我们假设，转录组和蛋白质组将在治疗小鼠中恢复正常，或者将识别出“糖尿病蛋白质组生物标记”，这将进一步加深我们对抗cd3作用机制的理解。这些研究将采用两种独特的尖端技术平台（eTag毛细管电泳分析和反相蛋白酸微阵列（RPPLM）分析）分别研究转录组和蛋白质组的一个子集。我们将利用斯坦福大学现有的独特资源来完成这些研究，包括与C. Garrison Fathman博士（T1D和基因组学方面的专业知识）、Garry Nolan博士（免疫学、荧光活化细胞分选或FACS和蛋白质组学方面的专业知识）和Robert Tibshirani博士（蛋白质组学和生物医学信息学方面的专业知识）合作。该团队将测试组合基因组和蛋白质组学分析的能力，以获得T1D发展中进展阶段的清晰和可重复的特征模式。这些试验将应用于4个相关的特定目的：(i)分析来自NOD. bdc2.5小鼠和NOD的pln的纯化CD4+ T细胞的多种信号通路。B10小鼠，在不同时间点处死，反映糖尿病前期和显性糖尿病状态；（ii）测定抗cd3治疗NOD小鼠的CD4+ T细胞中多种信号蛋白的激活状态，并比较未治疗和治疗小鼠的信号蛋白组；（iii）利用激光捕获显微解剖（LCM）分析胰岛浸润淋巴细胞和β细胞的信号蛋白组；（iv）确定外周血CD4+ T细胞是否反映了转录组和蛋白质组的类似变化。如果这些模式是相似的，那么这些试验可能为疾病进展和评估人类治疗反应提供可靠的替代T细胞标志物。这项工作完全符合美国国立卫生研究院的科学使命，并且仅针对NOD小鼠进行研究，NOD小鼠是目前最有用的T1D小鼠模型之一。我们的长期目标是使用同样的方法来研究来自人类糖尿病患者的血细胞，希望使我们能够对患者进行分类，并定义新的患者特异性或靶向特异性治疗模式。

DESCRIPTION (provided by applicant): The proposed studies seek to analyze an important subset of the transcriptome and proteome in mouse models of type I diabetes (T1D), specifically characterizing the transcriptome and proteome of pancreatic lymph node (PLN) cells, islet beta cells, and peripheral blood cells over time. We will then use a systems biology approach to perturb the system through treatment of mice with anti-CD3 monoclonal antibodies, which have been shown to reverse hyperglycemia in newly-diabetic mice, and are currently in clinical trials in human beings. We hypothesize that the transcriptome and proteome will revert to normal in treated mice, or will instead identify a "diabetes proteomic biosignature" that will further our understanding of the mechanisms of action of anti-CD3. These studies will employ two unique and cutting-edge technology platforms (eTag capillary electrophoresis assays, and reverse phase protein mysate microarray (RPPLM) assays) for studying a subset of the transcriptome and proteome, respectively. We will draw on unique resources existing at Stanford University to complete these studies, including collaborations with Dr. C. Garrison Fathman (expertise in T1D and genomics), Dr. Garry Nolan (expertise in immunology, fluorescence activated cell sorting or FACS, and proteomics) and Dr. Robert Tibshirani (expertise in proteomics and biomedical informatics). This team will test the ability of a combined genomic and proteomic analysis to obtain a clear and reproducible signature pattern of progressive stages in the development of T1D. These assays will be applied to 4 related specific aims: (i.) to analyze multiple signaling pathways in purified CD4+ T cells from the PLNs of NOD.BDC2.5 mice and NOD.B10 mice, sacrificed at different time points reflecting the prediabetic and overtly diabetic state; (ii.) to determine the activation status of multiple signaling proteins in CD4+ T cells isolated from NOD mice treated with anti-CD3, and to compare the signaling proteome between untreated and treated mice; (iii.) to use laser capture microdissection (LCM) to analyze the signaling proteome of islet-infiltrating lymphocytes and beta cells; and, (iv.) to determine whether peripheral blood CD4+ T cells reflect similar changes in the transcriptome and proteome. If these patterns are similar, then these assays may provide a reliable surrogate T cell marker for disease progression and for assessment of therapeutic response in humans. This work is soundly within the scientific mission of the NIH, and is solely aimed at studying NOD mice, one of the most useful mouse models for T1D that exists. Our long term goal is to use the same approach for studying blood cells derived from human diabetic patients, hopefully allowing us to categorize patients and to define novel patient-specific or target-specific therapeutic modalities.