Lysate Arrays for Studying the Diabetes Proteome

描述（由申请人提供）：拟定研究旨在分析I型糖尿病（T1 D）小鼠模型中转录组和蛋白质组的重要子集，特别是表征胰腺淋巴结（PLN）细胞、胰岛β细胞和外周血细胞随时间推移的转录组和蛋白质组。然后，我们将使用系统生物学方法，通过用抗CD 3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症，目前正在人类临床试验中。我们假设，转录组和蛋白质组将恢复到正常的治疗小鼠，或将改为确定一个“糖尿病蛋白质组生物签名”，这将进一步了解抗CD 3的作用机制。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质mysate微阵列（RPPLM）分析），分别用于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（T1 D和基因组学专业知识）、Garry Nolan博士（免疫学、荧光激活细胞分选或FACS和蛋白质组学专业知识）和Robert Tibshirani博士（蛋白质组学和生物医学信息学专业知识）。该团队将测试基因组和蛋白质组联合分析的能力，以获得T1 D发展进行阶段的清晰且可重复的特征模式。这些试验将应用于4个相关的具体目标：（i.）分析来自NOD.BDC2.5小鼠和NOD.B10小鼠的PLN的纯化的CD 4 + T细胞中的多种信号传导途径，所述小鼠在反映糖尿病前期和明显糖尿病状态的不同时间点处死;（ii.）确定从用抗CD 3处理的NOD小鼠分离的CD 4 + T细胞中多种信号传导蛋白的活化状态，并比较未处理和处理的小鼠之间的信号传导蛋白质组;（iii.）使用激光捕获显微切割（LCM）来分析胰岛浸润淋巴细胞和β细胞的信号传导蛋白质组;以及，（iv.）以确定外周血CD 4 + T细胞是否反映转录组和蛋白质组的类似变化。如果这些模式是相似的，那么这些检测可以提供一个可靠的替代T细胞标志物的疾病进展和评估人类的治疗反应。这项工作完全符合NIH的科学使命，并且仅旨在研究NOD小鼠，NOD小鼠是现有的T1 D最有用的小鼠模型之一。我们的长期目标是使用相同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目标特异性治疗方式。

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.