Aptamers for Analysis of Extracellular Vesicles

在研究项目中，我提出了细胞外囊泡（EVs）的亲和力分离，EVs的检测和计数以及EVs生物标志物的综合分析的新技术。ev是由许多细胞类型分泌到细胞外环境的小的膜封闭结构。这些颗粒包括小泡（外泌体，30- 150nm）和大泡（微泡，100 - 1000nm；凋亡小体，50 - 5000nm）。这些囊泡由细胞分泌，并在体内不同的液体中释放。电动汽车含有一层脂质双分子层，保护由核酸、蛋白质、脂质和代谢物组成的货物免受降解。电动汽车现在是生物科学的核心，因为它们似乎构成了一种新的细胞-细胞通讯系统。由于电动汽车的货物反映了细胞的精确状态，因此人们对利用电动汽车进行生物标志物分析非常感兴趣。ev是创伤性脑损伤（TBI）和创伤后应激障碍（PTSD）等疾病生物标志物的丰富来源。最近，人们认识到来自脑细胞的ev携带来自母细胞的蛋白质和核酸并穿过血脑屏障，可以在外周血和唾液中检测到，这为评估创伤后应激障碍和创伤后脑损伤期间神经元和胶质细胞的分子变化提供了新的机会。在接下来的5年里，我和我的研究团队计划提供两种新的技术来生产针对电动汽车的特异性亲和探针，即核酸适体。所得到的适体对源自脑细胞的外泌体和微泡具有可切换的结合和高选择性。两种电动汽车隔离技术（磁珠和柱为基础）也将开发和发表，为电动汽车研究界的利益。我们期望从人类样本中获得高回收率的分离ev，用于下游应用，如miRNA和蛋白质分析。我们将开发和共享至少两项基于毛细管电泳和质谱的电动汽车蛋白质和microrna综合分析技术。从这些方法获得的数据将纳入公共数据库。特别是，我们将开发一种基于适配体辅助脑ev分离和TBI/PTSD生物标志物分析的突破性诊断方法，以区分损伤状态。从长远来看，我计划对适体的选择、结构、功能和相关结合伙伴进行大规模的研究。我想在miRNA数据和生物信息学分析的支持下，利用蛋白质组学和代谢组学的高通量方法研究电动汽车。总的来说，我将讨论电动汽车的起源和最终目的地，分子多样性和生物学功能的基本问题。脑细胞源性ev生物检测技术的发展将为研究脑外伤、创伤后应激障碍和退行性脑疾病的基本过程的研究人员带来新的分析工具。

In the research program grant, I propose new technologies for the affinity isolation of extracellular vesicles (EVs), the detection and counting of EVs, and comprehensive analysis of EV biomarkers. EVs are small membrane-enclosed structures that are secreted into the extracellular milieu by many cell types. These particles include small (exosomes, 30-150 nm), and large (microvesicles, 100 - 1000 nm; apoptotic bodies, 50 - 5000 nm) vesicles. These vesicles are secreted from cells and released in different fluids of the body. EVs contain a lipid bilayer that protects the cargo, which can consist of nucleic acids, proteins, lipids and metabolites, from degradation. EVs are now central to biological sciences because they seem to constitute a new system of cell-cell communication. Since the cargo of EVs reflects the precise state of a cell, there has been significant interest in utilizing EVs in biomarker analysis. EVs are a plentiful source of biomarkers of traumatic brain injury (TBI) and posttraumatic stress disorder (PTSD) among other conditions. Recently, the recognition that EVs derived from brain cells that carry proteins and nucleic acid from their mother cells and cross the blood brain barrier can be detected in peripheral blood and saliva, offers a new opportunity to evaluate molecular changes in neurons and glia during PTSD and after TBI. Over the next 5 years, my research team and I plan to provide two novel technologies for the production of specific affinity probes for EVs, named nucleic acid aptamers. The resulting aptamers will have switchable binding and high selectivity to exosomes and microvesicles originating from brain cells. Two EV isolation technologies (magnetic bead and column - based) will be also developed and published, for the benefit of the EV research community. We expect to achieve high recovery of isolated EVs from human samples for downstream applications such as miRNA and protein assays. We will develop and share at least two technologies based on capillary electrophoresis and mass spectrometry for comprehensive analysis of proteins and microRNAs of EVs. The data obtained from these approaches will be integrated into public databases. In particular, we will develop a breakthrough diagnostic based on aptamer-assisted isolation of brain EVs and analysis of TBI/PTSD biomarkers to discriminate injury states. In the long-term, I plan to perform large-scale research on selection of aptamers, their structures, functions and related binding partners. I want to study EVs using high-throughput approaches in proteomics and metabolomics, supported by miRNA data and bioinformatic analysis. In general, I will address fundamental issues of EV origins and final destinations, molecular diversity and biological functions. The development of bioassays for brain cell-derived EVs will bring new analytical tools for researchers investigating fundamental processes of TBI, PTSD and degenerative brain disorders.