In vivo Cell-Specific Exosome Analysis

外泌体是直径约30-150 nm的小的单膜分泌细胞器 大多数细胞分泌的蛋白质。越来越多的人认识到，外泌体是细胞间通讯的重要部分，并且外泌体携带的货物将多种信号传导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以反映该特定细胞群的生理状态。 外泌体的大多数研究依赖于从体外培养的细胞或细胞簇中分离外泌体，或从生物流体（如血浆）的体内取样中大量分离外泌体。然而，不存在允许体内分离和检查可以追溯到特定起源细胞并且可以在同一生物体内随时间纵向取样的外来体的简单工具。 因此，我们几乎没有关于在生物体的生命过程中，外泌体及其从特定细胞释放的货物如何随着衰老而变化的信息，或者如果特定细胞类型响应增加的功能需求和/或损伤，外泌体货物如何变化的信息。 为了解决这些问题，我们已经产生了一种小鼠模型，该模型允许通过表达工程化的外泌体表面标记物对外泌体进行条件性和细胞选择性遗传标记。我们已经使用了一个cDNA盒，其中绿色荧光标签（pHluorin）的编码序列插入到四跨膜蛋白CD 63的第一个胞外结构域中，四跨膜蛋白CD 63高度富集在外泌体表面并在外泌体表面表达。我们已经将该盒插入Rosa 26基因座中，使其在CRE介导的上游转录终止序列（= R26-R26）的去除后以细胞特异性方式条件性表达。 StopLoxP-CD 63-pHluorin小鼠）。从这些小鼠的血浆中，使用识别pHluorin抗原的抗体，我们可以分离和捕获可以追溯到其起源细胞的外泌体。这种独特的小鼠模型代表了以细胞选择性方式在体内研究外泌体的现有方法的显著进步。 我们选择研究从胰腺癌细胞释放的外泌体，因为已知这些细胞随着衰老以及在不同的生理和病理条件下经历不同的功能和分子变化。在这个探索性的建议中，我们的目标是使用我们的模型系统来检查来自小鼠胰腺β细胞的外泌体货物是否以及如何在其一生中以及在代谢需求改变的情况下发生变化。在分子水平上，我们将把我们的原理验证研究集中在检查 图10.在PBMC和循环PBMC来源的外泌体中的RNA水平。 这些研究的结果将增加我们对外泌体是否准确反映胰腺癌细胞的年龄和功能状态以及外泌体及其货物是否可用作早期检测细胞功能障碍和对治疗反应的诊断工具的知识。

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.