Defining Islet Heterogeneity Using Single Islet Transcriptomics

使用单胰岛转录组学定义胰岛异质性

基本信息

批准号：
8812982
负责人：
MARTHA CAMPBELL-THOMPSON
金额：
$ 159.77万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-19 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8812982
关键词：
Address Animal Model Animals Antibodies Area Autoantibodies Beta Cell Biological CD3 Antigens Cell Death Cell Survival Cells Cellular Stress Collaborations Complex Critical Pathways Crude Extracts Cryoultramicrotomy Data Development Diabetes Mellitus Diabetes autoantibodies Disease Disease Marker Fluorescence Microscopy Freezing Functional disorder Gene Expression Gene Expression Profile Gene Expression Profiling Genes Head Heterogeneity Human Immune Immune System and Related Disorders Immunofluorescence Immunologic Individual Inflammation Insulin Insulin-Dependent Diabetes Mellitus Lasers Leukocytes Measures Metabolic stress Methods Microdissection Microscope Mission Molecular Molecular Profiling Organ Organ Donor Pancreas Paraffin Embedding Pathway interactions Pattern Phenotype Process Process Measure Protocols documentation RNA Reporting Research Research Personnel Serum Staging Staining method Stains Stress Surveys System T-Lymphocyte Tail Technology Therapeutic Time Tissues Viral Markers Virus Diseases base cell injury diabetic insight interest islet laser capture microdissection new technology non-diabetic novel novel strategies novel therapeutics prevent public health relevance research and development tool transcriptomics type I diabetic

项目摘要

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.

描述（由申请人提供）：我们的研究集中于检查单胰岛基因表达谱（转录组），以确定对人β细胞死亡至关重要的途径。在初步研究中，我们开发了新的方法和方案，这些方法和方案允许从人类胰腺的冷冻切除术，提取RNA并获得全面的转录组来激光捕获单个胰岛。我们对HIRN-CBD联盟的建议基于使用和扩展这些验证的方案，以更深入地了解胰岛异质性和来自有或没有糖尿病患者，或没有血清自身抗体的个体的个体胰岛基因表达模式的差异（AAB）。基于有或没有AAB的非糖尿病个体的胰岛中的初步数据，我们假设胰岛在胰岛素的早期阶段之前会形成“细胞应激”的基因表达特征。胰岛基因表达特征的早期异常的完全表征可以指出疾病的新机制和新的治疗策略，这些机制靶向胰岛异常，以延迟或防止β细胞死亡。使用数千个表达基因的综合基因表达分析将非常详细地查看特定的途径和过程。我们的建议具有以下特定目的：特定目的1。在对来自有或没有自身抗体（AAB）的器官捐献者的胰岛和有或没有糖尿病的胰岛的胰岛调查中，表征单个胰岛的转录组。这将提供对胰岛基因表达及其异质性的无偏调查。特定目的2。表征通过快速免疫激光捕获显微解剖选择的单个胰岛的转录组，以在存在或不存在胰岛素阳性β细胞以及存在或不存在CD3 T细胞的情况下。在此目标中，我们将从β细胞和胰岛炎的多种荧光污渍中选择的胰岛获得表达谱。我们将开发用于使用快速免疫荧光的方案，该方案基于它们的染色模式，可提供高质量的RNA，然后在跨串行切片的跨部门直接进行微解剖胰岛。特定目的3。要表征选择用于染色病毒标记模式的单个小岛的转录组存在和代谢压力。在此目标中，我们将使用新获得的能力进行多重免疫荧光来定义具有新型复杂表型的胰岛，以表明由于病毒感染或代谢应激而导致的压力。在此目标中，表型将非常复杂，我们将染色一个组织截面，然后从相邻的串行组织切片中捕获单个感兴趣的小岛。使用孤立胰岛的机械研究将遵循所有特定的目的，因为发现了细胞破坏或存活的新途径。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（1）

Organ donor specimens: What can they tell us about type 1 diabetes?

器官捐献者标本：它们能告诉我们有关 1 型糖尿病的哪些信息？

DOI：
10.1111/pedi.12286
发表时间：
2015-08-01
期刊：
PEDIATRIC DIABETES
影响因子：
3.4
作者：
Campbell-Thompson, Martha
通讯作者：
Campbell-Thompson, Martha

Alpha cell receptor for advanced glycation end products associate with glucagon expression in type 1 diabetes.

DOI：
10.1038/s41598-023-39243-x
发表时间：
2023-08-09
期刊：
SCIENTIFIC REPORTS
影响因子：
4.6
作者：
Leung, Sherman S.;Lenchik, Nataliya;Mathews, Clayton;Pugliese, Alberto;McCarthy, Domenica A.;Le Bagge, Selena;Ewing, Adam;Harris, Mark;Radford, Kristen J.;Borg, Danielle J.;Gerling, Ivan;Forbes, Josephine M.
通讯作者：
Forbes, Josephine M.

Genetic predisposition in the 2'-5'A pathway in the development of type 1 diabetes: potential contribution to dysregulation of innate antiviral immunity.

DOI：
10.1007/s00125-021-05469-5
发表时间：
2021-08
期刊：
Diabetologia
影响因子：
8.2
作者：
Pedersen K;Haupt-Jorgensen M;Krogvold L;Kaur S;Gerling IC;Pociot F;Dahl-Jørgensen K;Buschard K
通讯作者：
Buschard K

The IGFBP3/TMEM219 pathway regulates beta cell homeostasis.

DOI：
10.1038/s41467-022-28360-2
发表时间：
2022-02-03
期刊：
Nature communications
影响因子：
16.6
作者：
D'Addio F;Maestroni A;Assi E;Ben Nasr M;Amabile G;Usuelli V;Loretelli C;Bertuzzi F;Antonioli B;Cardarelli F;El Essawy B;Solini A;Gerling IC;Bianchi C;Becchi G;Mazzucchelli S;Corradi D;Fadini GP;Foschi D;Markmann JF;Orsi E;Škrha J Jr;Camboni MG;Abdi R;James Shapiro AM;Folli F;Ludvigsson J;Del Prato S;Zuccotti G;Fiorina P
通讯作者：
Fiorina P

Insulitis and β-Cell Mass in the Natural History of Type 1 Diabetes.

DOI：
10.2337/db15-0779
发表时间：
2016-03
期刊：
Diabetes
影响因子：
7.7
作者：
Campbell-Thompson M;Fu A;Kaddis JS;Wasserfall C;Schatz DA;Pugliese A;Atkinson MA
通讯作者：
Atkinson MA