In vivo Cell-Specific Exosome Analysis

体内细胞特异性外泌体分析

基本信息

批准号：
10231459
负责人：
Mehboob A Hussain
金额：
$ 19.5万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10231459
关键词：
Address Age Aging Antibodies Antidiabetic Drugs Antigens Back Benchmarking Beta Cell Biological Biological Models Blood Circulation Caliber Cell physiology Cells Chronology Code Complement Complementary DNA Cultured Cells Diabetes Mellitus Diagnostic Dietary Intervention Early Diagnosis Excision Exhibits Extracellular Domain Foundations Functional disorder Genetic Genetic Transcription In Vitro Injury Insulin Resistance Intervention Knowledge Label Life Cycle Stages Liquid substance Mediating Membrane Metabolic Metabolic stress MicroRNAs Modeling Molecular Mus Organelles Organism Outcome Outcome Study PHluorin Pathologic Pharmacology Physiological Plasma Population Property RNA Sampling Signal Transduction Structure of beta Cell of islet Surface Testing Therapeutic Effect Therapeutic Intervention Time base cell age cell type cohort diet-induced obesity dietary engineered exosomes exenatide exosome glycemic control in vivo insulin secretion intercellular communication islet liquid biopsy molecular marker mouse model novel recombinase-mediated cassette exchange response selective expression sex tool treatment response

项目摘要

Exosomes are small single-membrane secreted organelles of approximately 30-150 nm in diameter that are secreted by most, if not all cells. There is a growing appreciation that exosomes are an important part of inter-cellular communication and that cargo carried by exosomes transmit a variety of signaling properties from their cell of origin to cells that receive exosome cargo. Further, the exosome cargo released from a specific cell-type may reflect the physiologic state of that particular cell population. Most studies on exosomes rely on isolating exosomes from cultured cells or cell clusters in vitro, or bulk exosome isolation from in vivo sampling of biological fluids (such as plasma). However, there exist no simple tools that allow to in vivo isolate and examine exosomes that can be traced back to a specific cell of origin and which can be sampled longitudinally over time within the same organism. Thus, we have little information on how exosomes and their cargo released from a certain cell changes with ageing during the course of the life of an organism or how the exosome cargo may change if a particular cell-type responds to increased functional demand and/or injury. To address these questions, we have generated a mouse model that permits conditional and cell-selective genetic labeling of exosomes through expression of an engineered exosome surface marker. We have used a cDNA cassette in which the coding sequence of a green-fluorescent tag (pHluorin) is inserted into the first extracellular domain of the tetraspanin CD63, which is highly enriched in and expressed on the surface of exosomes. We have inserted this cassette into the Rosa26 locus allowing it to be conditionally expressed in a cell-specific manner after CRE-mediated removal of upstream transcriptional stop sequences (= R26- StopLoxP-CD63-pHluorin mouse). From plasma of these mice, using an antibody that recognizes pHluorin antigen, we can isolate and capture exosomes that can be traced back to their cell of origin. This unique mouse model represents a significant advance over existing means to study exosomes in vivo in a cell-selective manner. We have chosen to study exosomes released from pancreatic ß-cells, as these cells are known to undergo distinct functional and molecular changes with ageing and under different physiologic and pathologic conditions. In this exploratory proposal we aim to use our model system to examine whether and how the exosome cargo from pancreatic ß-cells in mice changes over their lifetime and during circumstances of altered metabolic demand. At the molecular level we will focus our proof-of-principle studies on examining changes in RNA levels in ß-cells and in circulating ß-cell-derived exosomes. The outcomes of these studies will increase our knowledge on whether exosomes accurately reflect the age and functional state of the pancreatic ß-cell and whether exosomes and their cargo could be utilized as diagnostic tools for early detection of cellular dysfunction and response to treatment.

外泌体是直径约为 30-150 nm 的小型单膜分泌细胞器人们越来越认识到外泌体是细胞间通讯的重要组成部分，并且外泌体携带的货物将多种信号特性从其来源细胞传递到接收外泌体货物的细胞。此外，从特定细胞类型释放的外泌体货物可能反映该特定细胞群的生理状态，大多数外泌体研究依赖于从体外培养的细胞或细胞簇中分离外泌体，或从体内取样中分离大量外泌体。然而，没有简单的工具可以在体内分离和检查外泌体，这些外泌体可以追溯到特定的起源细胞，并且可以在同一生物体内随时间纵向采样。我们对某些细胞释放的外泌体及其货物在生物体生命过程中如何随着衰老而变化，或者如果特定细胞类型对功能需求增加和/或损伤做出反应，外泌体货物可能如何变化的信息知之甚少。为了解决这些问题，我们有生成了一个小鼠模型，该模型允许通过表达工程外泌体表面标记来对外泌体进行条件性和细胞选择性遗传标记。我们使用了 cDNA 盒，其中绿色荧光标签 (pHluorin) 的编码序列被插入到第一个细胞外。四跨膜蛋白 CD63 的结构域在外泌体表面高度富集和表达，我们已将该盒插入到 Rosa26 基因座中，使其能够在外泌体中条件性表达。 CRE介导的上游转录终止序列去除后的细胞特异性方式（= R26- 使用识别 pHluorin 抗原的抗体，我们可以从这些小鼠的血浆中分离和捕获可追溯到其细胞来源的外泌体，这种独特的小鼠模型代表了现有方法的重大进步。以细胞选择性的方式研究体内外泌体，我们选择研究胰腺β细胞释放的外泌体，因为已知这些细胞会随着衰老和衰老而发生明显的功能和分子变化。在这个探索性建议中，我们的目标是使用我们的模型系统来检查小鼠胰腺β细胞的外泌体在其一生中以及在代谢需求的情况下是否以及如何变化。将我们的原理验证研究重点放在检查变化上这些研究的结果将增加我们对外泌体是否准确反映胰腺β细胞的年龄和功能状态以及外泌体及其货物是否可以利用的认识。作为早期检测细胞功能障碍和治疗反应的诊断工具。