Department of Craniofacial Biology, College of Dental Medicine, Medical University of South Carolina, Charleston, South Carolina, United States of America.
PLoS One. 2010 Jan 5;5(1):e8577. doi: 10.1371/journal.pone.0008577.
Exosomes, derived from endocytic membrane vesicles are thought to participate in cell-cell communication and protein and RNA delivery. They are ubiquitous in most body fluids (breast milk, saliva, blood, urine, malignant ascites, amniotic, bronchoalveolar lavage, and synovial fluids). In particular, exosomes secreted in human saliva contain proteins and nucleic acids that could be exploited for diagnostic purposes. To investigate this potential use, we isolated exosomes from human saliva and characterized their structural and transcriptome contents.
Exosomes were purified by differential ultracentrifugation and identified by immunoelectron microscopy (EM), flow cytometry, and Western blot with CD63 and Alix antibodies. We then described the morphology, shape, size distribution, and density using atomic force microscopy (AFM). Microarray analysis revealed that 509 mRNA core transcripts are relatively stable and present in the exosomes. Exosomal mRNA stability was determined by detergent lysis with RNase A treatment. In vitro, fluorescently labeled saliva exosomes could communicate with human keratinocytes, transferring their genetic information to human oral keratinocytes to alter gene expression at a new location.
Our findings are consistent with the hypothesis that exosomes shuttle RNA between cells and that the RNAs present in the exosomes may be a possible resource for disease diagnostics.
外泌体来源于内吞膜囊泡，被认为参与细胞间通讯以及蛋白质和 RNA 的传递。它们广泛存在于大多数体液中（母乳、唾液、血液、尿液、恶性腹水、羊水、支气管肺泡灌洗液和滑液）。特别是，人唾液中分泌的外泌体含有可用于诊断目的的蛋白质和核酸。为了研究这种潜在用途，我们从人唾液中分离出外泌体，并对其结构和转录组内容进行了表征。
通过差速超速离心法纯化外泌体，并通过免疫电子显微镜（EM）、流式细胞术和 CD63 和 Alix 抗体的 Western blot 进行鉴定。然后，我们使用原子力显微镜（AFM）描述了外泌体的形态、形状、大小分布和密度。微阵列分析显示，509 个 mRNA 核心转录物相对稳定存在于外泌体中。通过去污剂裂解并用 RNase A 处理来确定外泌体 mRNA 的稳定性。在体外，荧光标记的唾液外泌体可以与人类角质形成细胞相互作用，将其遗传信息传递给人类口腔角质形成细胞，从而在新的位置改变基因表达。
我们的发现与外泌体在细胞间传递 RNA 的假说一致，并且外泌体中存在的 RNA 可能是疾病诊断的一种潜在资源。
