In vivo Cell-Specific Exosome Analysis

Exosome是一种小的单膜分泌细胞器，直径约30-150 nm 即使不是全部，也是由大多数细胞分泌的。人们越来越认识到，外切体是细胞间通讯的重要组成部分，外切体携带的货物将各种信号特性从其起源的细胞传递到接收外切体货物的细胞。此外，从特定细胞类型释放的外切体货物可以反映该特定细胞群体的生理状态。大多数对外切体的研究依赖于从体外培养的细胞或细胞团中分离外切体，或从生物体液(如血浆)的体内采样中分离大量外切体。然而，目前还没有简单的工具可以在体内分离和检查可追溯到特定起源细胞并可在同一生物体内随时间纵向采样的外体。因此，我们几乎没有关于特定细胞释放的外切体及其货物在生物体的生命过程中如何随着年龄的变化而变化的信息，或者如果特定细胞类型对增加的功能需求和/或伤害做出反应时外切体货物可能如何变化的信息。为了解决这些问题，我们建立了一个小鼠模型，允许通过表达一个工程的外切体表面标记来对外切体进行有条件的和细胞选择性的遗传标记。我们已经使用了一个插入绿色荧光标记(PHluorin)编码序列到Tetraspanin CD63的第一个胞外区的cDNA盒，Tetraspanin CD63在外切体表面高度丰富和表达。我们已经将这个盒式磁带插入到rosa26基因座中，使其在Cre介导的上游转录停止序列(=R26-)被移除后，以细胞特异性的方式有条件地表达。 StopLoxP-CD63-pHluorin小鼠)。从这些小鼠的血浆中，使用一种识别PHLOORIN抗原的抗体，我们可以分离和捕获可以追溯到它们起源细胞的外体。这一独特的小鼠模型代表着对现有方法的重大进步，即以细胞选择性的方式在体内研究外切体。我们选择研究胰腺细胞释放的外切体，因为已知这些细胞在不同的生理和病理条件下，随着年龄的增长会经历不同的功能和分子变化。在这个探索性的提案中，我们的目标是使用我们的模型系统来检查小鼠胰腺细胞的外切体货物在它们的一生中以及在代谢需求改变的情况下是否以及如何变化。在分子水平上，我们将把我们的原则证明研究的重点放在检查 ？细胞和由？细胞衍生的外体循环中的RNA水平。这些研究的结果将增加我们对外切体是否准确地反映胰腺细胞的年龄和功能状态，以及外切体及其货物是否可以作为早期发现细胞功能障碍和治疗反应的诊断工具的认识。

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.