Defining Islet Heterogeneity Using Single Islet Transcriptomics

描述(由申请人提供)：我们的研究集中在单个胰岛基因表达谱(转录本)的检查，以确定对人类β细胞死亡和生存至关重要的途径。在初步研究中，我们开发了新的方法和方案，允许激光从人胰腺的冰冻切片中捕获单个胰岛，提取RNA，并获得全面的转录本。我们向Hirn-CBDS联盟提出的建议是基于使用和扩展这些成熟的方案，以更深入地了解胰岛的异质性和个体胰岛基因表达模式的差异，这些个体来自患有和不患有糖尿病以及有或没有血清自身抗体(AAB)的个体。基于患有或不患有AAB的非糖尿病患者的胰岛的初步数据，我们假设胰岛在胰岛炎症的早期阶段之前就形成了“细胞应激”的基因表达特征。对胰岛基因表达特征的早期异常的充分表征可能指向新的疾病机制和新的治疗策略，针对胰岛异常来延迟或防止β细胞死亡。使用对数千个表达的基因进行全面的基因表达谱分析将允许非常详细地查看特定的途径和过程。我们的建议有以下具体目标：具体目标1.对来自胰腺不同区域的胰岛和来自器官捐赠者的有或无自身抗体(AAB)以及有或没有糖尿病的胰岛进行调查，以确定单个胰岛的转录本。这将为胰岛基因的表达及其异质性提供一个公正的调查。具体目的2.通过快速免疫激光捕获显微切割技术选择单个胰岛的转录本，以确定是否存在胰岛素阳性的β细胞和存在或不存在CD3T细胞。在这个目标中，我们将从胰岛中获得表达谱，这些胰岛是根据β细胞和胰岛素炎的多种荧光染色而选择的。我们将开发使用快速免疫荧光的方案，提供高质量的RNA，然后根据胰岛的染色模式，跨连续切片直接显微切割胰岛。特定目的3.确定用于病毒标记染色模式的单个胰岛的转录本的特征 存在和新陈代谢压力。在这个目标中，我们将利用我们新获得的进行多重免疫荧光的能力来定义具有新的复杂表型的胰岛，这些表型表明由于病毒感染或代谢应激所致的应激。在这个目标中，表型将是高度复杂的，我们将对一个组织切片进行染色，然后从相邻的连续组织切片中捕获感兴趣的单个胰岛。随着细胞破坏或存活的新途径被发现，使用孤立的胰岛的机制研究将遵循所有特定的目标。

DESCRIPTION (provided by applicant): Our studies are focused on examination of single islet gene expression profiles (transcriptomes) to determine pathways critical to human beta cell death and survival. In preliminary studies we have developed new approaches and protocols that allow laser-capture of individual islets from cryosections of human pancreas, extract the RNA, and obtain comprehensive transcriptomes. Our proposal to the HIRN-CBDS consortium is based on using and expanding these proven protocols to gain deeper insights into islet heterogeneity and differences in gene expression patterns of individual islets from individuals with and without diabetes and with or without serum autoantibodies (AAB). Based on preliminary data in islets from non-diabetic individuals with or without AAB, we hypothesize that islets develop a gene expression signature of "cellular stress" before the early stages of insuliti. A full characterization of early abnormalities in islet gene expression signatures could point to novel mechanisms of disease and new therapeutic strategies that target islet abnormalities to delay or prevent beta cell death. The use of comprehensive gene expression profiling of thousands of expressed genes will allow a very detailed look at specific pathways and processes. Our proposal has the following specific aims: Specific Aim 1. To characterize transcriptomes of individual islets in a survey of islets from different regions of the pancreas an from organ donors with or without autoantibodies (AAB) and with or without diabetes. This will provide an unbiased survey of islet gene expression and its heterogeneity. Specific Aim 2. To characterize transcriptomes of individual islets selected by rapid immuno-laser capture microdissection for the presence or absence of insulin-positive beta cells and presence or absence of CD3 T-cells. In this aim we will obtain expression profiles from islets that have been selected based on multiple fluorescent stains for beta cells and insulitis. We will develop protocols for using rapid immunofluorescence that provide high quality RNA and then directly microdissect islets, across serial sections, based on their staining patterns. Specific Aim 3. To characterize transcriptomes of individual islets selected for staining patterns of markers of viral presence and metabolic stress. In this aim we will use our newly acquired ability to conduct multiplexed immunofluorescence to define islets with novel complex phenotypes indicative of stress due to viral infections or metabolic stress. In this aim phenotypes will be highly complex and we will stain one tissue section and then capture individual islets of interest from adjacent serial tissue sections. Mechanistic studies using isolated islets will follow from all specific aim as new pathways of cellular destruction or survival are discovered.