Expression data from Human Saliva Exosomes. Homo sapiens

Exosomes are molecular entities derived from membrane vesicles of endocytic origin secreted by most cell types. These vesicles are implicated in cell-to-cell communication, deliver proteins and mRNA molecules between cells. Recent studies have shown that exosomes are found in body fluids such as saliva, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluids and breast milk. Exosomes secreted through human saliva contain mRNA may potentially be useful for diagnostic purposes. Although the exact protective mechanism of saliva RNA is a topic of debate, the consensus is that the enrichment of mRNAs in these nano-vesicles in one of the features of the biomarker discoveries. Our aim was to determine if exosomes are present in human saliva and to nano-characterize their transcriptomic content. Exosomes were purified by differential ultracentrifugation, identified by immunoelectron microscopy, flow cytometry and western blot using a CD-63 antibody. Atomic force microscopy studies revealed ultra structural analysis of both size and density of exosomes. Microarray analysis revealed the presence of 590 mRNA core transcripts are relatively stable inside the exosomes, which can be of saliva mRNA biomarkers. Exosomal mRNA stability was determined by detergent lyses with treatment of RNase. Under in vitro conditions fluorescent dye labeled saliva exosomes were able to communicate between human oral keratinocytes studied by using fluorescence microscopy. The RNA from saliva exosomes can transfer their genetic information to human oral keratinocytes and alters gene expression in the new location. Together, these results suggest that saliva is involved in mRNA trafficking via exosomes, and provides a mechanism for cargoing passenger mRNAs. Our findings are consistent with proposal that exosomes can shuttle RNAs between cells and mRNA is protected inside these vesicles may be a possible resource for biomarker discovery. Keywords: Human saliva, exosomes, mRNA profiling, gene expression, disease diagnosis Overall design: Human saliva exosomes were purified through differential centrifugation followed by RNA extraction and hybridization on Affymetrix microarrays. We were able to obtain normal human subjects saliva which are pooled and subjected to ultracentrifugation. The protocol was approved by UCLA Institutional review board. 1 ml of saliva exosomes were used to extract RNA followed by two rounds of amplification by Actorus Amp kit. The amplified RNA was biotin labled and hybridized with Affymetrix protocol.

外泌体（exosomes）是大多数细胞类型分泌的、源自内吞途径膜囊泡的分子实体。这类囊泡参与细胞间通讯，可在细胞间递送蛋白质与信使核糖核酸（mRNA）分子。
近期研究表明，外泌体存在于唾液、血液、尿液、羊水、恶性腹水、支气管肺泡灌洗液、滑液以及母乳等体液中。人体唾液分泌的外泌体所含的mRNA，有望应用于疾病诊断领域。尽管唾液RNA的确切保护机制尚存学术争议，但学界共识认为，这类纳米囊泡中富集的mRNA是生物标志物发现的重要特征之一。
本研究旨在确认人体唾液中是否存在外泌体，并对其转录组内容进行纳米尺度表征。
研究采用差速超速离心法纯化外泌体，通过免疫电子显微镜、流式细胞术以及使用CD63抗体的蛋白质印迹法对外泌体进行鉴定。原子力显微镜研究实现了外泌体尺寸与密度的超微结构分析。
基因芯片分析显示，唾液外泌体中存在590种相对稳定的核心mRNA转录本，可作为唾液mRNA生物标志物的候选来源。
外泌体mRNA的稳定性通过核糖核酸酶处理结合去污剂裂解的方式进行测定。
体外实验中，经荧光染料标记的唾液外泌体可在人口腔角质形成细胞间进行通讯，该结果通过荧光显微镜得以验证。
唾液外泌体携带的RNA可将遗传信息传递至人口腔角质形成细胞，并在新宿主细胞中改变基因表达模式。
综上，上述结果表明唾液可通过外泌体介导mRNA的跨细胞运输，为功能性mRNA的囊泡包裹运输提供了机制支撑。本研究结果与“外泌体可在细胞间穿梭转运RNA，且囊泡内部的mRNA受到保护”这一假说相符，该类囊泡或可成为生物标志物发现的潜在资源。
关键词：人体唾液、外泌体、mRNA谱分析、基因表达、疾病诊断
整体实验设计：通过差速离心法纯化人体唾液外泌体，随后提取RNA并在Affymetrix基因芯片上进行杂交。我们收集了健康受试者的混合唾液样本，经超速离心处理。本实验方案已获得加州大学洛杉矶分校（UCLA）机构审查委员会批准。取1毫升唾液外泌体用于RNA提取，随后通过Actorus Amp试剂盒进行两轮扩增。扩增后的RNA经生物素标记，按照Affymetrix标准实验流程进行芯片杂交。