Lysate Arrays for Studying the Diabetes Proteome

描述(申请人提供)：拟议的研究试图分析I型糖尿病(T1D)小鼠模型中转录和蛋白质组的一个重要子集，具体描述胰腺淋巴结(PLN)细胞、胰岛β细胞和外周血细胞随时间的转录和蛋白质组。然后，我们将使用系统生物学的方法，通过用抗CD3单抗治疗小鼠来扰乱系统，这种单抗已经被证明可以逆转新糖尿病小鼠的高血糖，目前正在人类的临床试验中。我们假设，在接受治疗的小鼠中，转录组和蛋白质组将恢复正常，或者相反，将识别一个“糖尿病蛋白质组生物特征”，这将进一步加深我们对抗CD3作用机制的理解。这些研究将使用两个独特和尖端的技术平台(eTag毛细管电泳法和反相蛋白质肉芽组织微阵列(RPPLM)分析)，分别研究转录组和蛋白质组的子集。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(在T1D和基因组学方面的专业知识)、Garry Nolan博士(在免疫学、荧光激活细胞分类或FACS和蛋白质组学方面的专业知识)和Robert Tibishani博士(在蛋白质组学和生物医学信息学方面的专业知识)的合作。该团队将测试联合基因组和蛋白质组分析的能力，以获得T1D发育过程中进展阶段的清晰和可重现的签名模式。这些分析将适用于4个相关的具体目标：(I)分析NOD.BDC2.5小鼠和NOD.B10小鼠PLN中纯化的CD4+T细胞中的多种信号通路，在不同的时间点处死，以反映糖尿病前期和明显的糖尿病状态；检测抗CD3抗体处理的NOD小鼠分离的CD4+T细胞中多种信号蛋白的激活状态，并比较处理前后小鼠的信号蛋白质组；用激光捕获显微切割(LCM)技术分析胰岛浸润性淋巴细胞和胰岛β细胞的信号蛋白质组；以确定外周血中的CD4+T细胞是否反映了类似的转录组和蛋白质组的变化。如果这些模式相似，那么这些检测可能为疾病进展和人类治疗反应的评估提供可靠的替代T细胞标记物。这项工作完全在美国国立卫生研究院的科学任务范围内，仅旨在研究NOD小鼠，这是目前存在的T1D最有用的小鼠模型之一。我们的长期目标是使用同样的方法来研究人类糖尿病患者的血细胞，希望能让我们对患者进行分类，并定义新的针对患者的或针对特定目标的治疗方式。

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.