外泌体分离及其蛋白质组学分析的研究进展

外泌体是一种由细胞分泌的膜性囊泡小体,直径通常为30~100 nm,密度为1.10~1.18 kg/L。外泌体广泛存在于各种体液中,可携带脂类、蛋白质、信使RNAs（mRNAs）、microRNAs（miRNAs）、非编码RNAs（ncRNAs）等多种重要的生物功能分子。许多证据表明,外泌体形成了一种特殊的细胞间信息传递系统,不仅影响细胞的生理状态,而且与多种疾病的发生与发展密切相关。该文简要综述了近年来在外泌体的分离和纯化方面取得的研究进展,并对蛋白质组学技术在外泌体分析中的应用进行了概述。此外,还对外泌体蛋白质组学研究的发展前景进行了展望。     

基于核酸适体的外泌体分子识别研究进展

自研究者证实外泌体承担了细胞外RNA等物质的运输功能以来, 关于外泌体来源与功能的研究一直备受关注. 近年来外泌体被发现具有作为疾病生物标志物的潜力, 使得拥有特定表面蛋白以及特定装载物的外泌体成为分析化学领域有价值的检测对象. 从化学本质角度来说, 外泌体的获取与分析需要依赖特异性的分子识别过程. 核酸适体作为分子识别单元, 因其特异性强、 亲和力高、 生物活性稳定、 易于合成和保存、 而且其序列和结构上具有可编程性, 易于设计和修饰, 已成功地用在外泌体相关的生物传感体系中. 本文从外泌体的化学组成及其具有生理、 病理意义的组分出发, 从外泌体通用生物标志物识别、癌细胞来源外泌体的检测及外泌体蛋白谱的分析这3个方面综述了以核酸适体作为分子识别单元在外泌体分析领域的代表性工作, 总结了现有的靶向外泌体的核酸适体序列信息以及应用场景, 阐述了利用化学合成与修饰以及DNA自组装等化学调控手段增强核酸适体分子识别性能的最新进展, 并从适用于外泌体分子识别的核酸适体的筛选以及化学修饰的角度, 对未来的研究方向进行了展望.     

血清外泌体的蛋白质组分析及其在骨质疏松中的应用

外泌体是细胞分泌的微小具膜囊泡，作为重要媒介参与细胞间的信号传递，已在疾病的诊断和治疗中发挥独特作用。骨质疏松症是全身性骨代谢疾病，容易引起骨密度减少并导致骨折，在老年人群中发病率很高，目前急需发展特异性体液诊断技术。本论文采用超速离心的方法，对血清中的外泌体进行了分离富集和表征，并采用液相色谱-质谱进行了外泌体蛋白质组学分析，共鉴定到了179个外泌体蛋白质，主要参与防御响应和免疫应答等生物过程。针对来自正常对照组、骨量减少组和骨质疏松组的血清样本，分离富集其中的外泌体，通过免标记定量蛋白质组学分析，分别鉴定到188、224和185个蛋白质。定量分析显示17个蛋白质的表达量在骨质疏松组和骨量减少组有显著改变（p<0.05），包括Integrin β 3、Integrin α 2 β 1、Talin 1和Gelsolin等，说明人体骨质在衰变过程中发生了系统性变化，并体现在血清外泌体中。该研究可为骨质疏松研究提供潜在的分子标志物，有助于阐明其病变机制。     

外泌体在糖尿病治疗中的研究进展

糖尿病（DMs）是威胁人类健康的严重代谢性疾病,其发病机制尚未明确,致病原因颇多。现已有多种糖尿病治疗方案,如注射外源胰岛素、植物源性天然产物、干细胞疗法等,但均存在难以改善胰岛β细胞、生物利用度差、免疫排斥等问题,因此亟待开发更加安全有效的治疗方案。外泌体（Exosomes）作为一种细胞分泌的囊泡,含有细胞内所有重要物质,且参与细胞间信息与物质传递,随着间充质干细胞等糖尿病有效治疗方案的逐步问世,同时包含多类同类物质的外泌体在糖尿病治疗中的潜力也逐步凸显,其可以通过多种途径同时降糖的作用被报道。本文综述了近十几年外泌体在Ⅰ型糖尿病（T1DM）与Ⅱ型糖尿病（T2DM）中的治疗方案,并介绍了其分子机制与具体治疗作用,以便为促进外泌体治疗策略在临床中的落实和应用提供参考。     

外泌体分离与纯化技术研究进展

外泌体是所有真核细胞分泌到细胞外的直径介于30～150 nm的一种膜性纳米囊泡,参与细胞间生物信号的传递。大量实验证据表明,外泌体参与多种生物功能并发挥重要作用,包括蛋白质、RNA和脂质等生物分子的转移及多种疾病生理和病理过程的调节,被认为是疾病诊断、治疗和预后的重要的生物标志物和药物载体,因此发展简单、高效、经济的外泌体分离与纯化技术将有助于疾病的早期诊断和精准治疗。目前,利用外泌体的物理化学和生物化学特性已开发出多种分离外泌体的技术,但仍缺乏标准化和规模化临床级外泌体的分离方法,从而限制了其临床应用。另外,对分离出的外泌体的特征、纯度和数量的鉴定是判断外泌体分离纯化方法优劣的重要指标。本文综述了外泌体分离与纯化技术以及鉴定方法的研究进展,主要讨论分离技术的机制、性能、挑战和前景以及外泌体的鉴定方法,以期为外泌体的分离纯化提供新的思路和解决策略。     

血清和血清外泌体的蛋白质组分析及其在肝内胆管癌中的应用

外泌体是由各种类型细胞在正常或非正常生理情况下分泌释放至细胞外且携带多种生物活性分子的细胞外囊泡,在细胞间通讯和免疫应答等生物过程中发挥着重要作用。肝内胆管癌是一种胆道上皮恶性肿瘤,早期无明显临床症状且生存率较低,目前常用的诊断手段包括依赖于影像设备的诊断方式和灵敏度及特异性较低的诊断标志物等,这些手段的不足对发展新的特异性标志物提出了需求。该文对血清中的外泌体进行了分离和表征,并采用液相色谱-质谱技术针对健康组与肝内胆管癌患者组的血清样本和血清外泌体样本进行了无标记定量蛋白质组学分析,分别从两种类型样本中鉴定并筛选到271和430种可信蛋白质。基于血清样本和血清外泌体样本的可信蛋白质定量表达值进行多维统计分析都能将健康组与肝内胆管癌患者组良好地区分开。对血清样本中鉴定到的蛋白质进行差异蛋白质筛选,肝内胆管癌患者组相对于健康组有15个上调和8个下调蛋白质;对血清外泌体样本中鉴定到的蛋白质进行差异蛋白质筛选,肝内胆管癌患者组相对于健康组有33个上调和18个下调蛋白质;基于两种样本筛选到的差异蛋白质中仅有4个是重复的,且基于血清外泌体样本的51个差异蛋白质中有35个蛋白质属于外泌体蛋白质数据库。针对差异蛋白质进行生物学信息分析,与差异蛋白质相关的分子功能、生物过程和信号通路主要涉及天然免疫反应、炎症反应和凝血等过程。该研究为发现肝内胆管癌的潜在生物标志物和探究肝内胆管癌的发生、发展和转移等过程提供了参考和借鉴价值。此外,通过比较研究发现血清外泌体样本能够获得较多的差异蛋白质和生物学信息,证明了外泌体作为组学分析样本的价值和应用潜力。     

细胞膜锚定DNA四面体传感器实时监测外泌体的分泌

外泌体是细胞主动分泌的一种纳米级双层膜结构的小囊泡,能够直接反映分泌细胞的生理和功能状态,进行细胞间的物质运输和信息通讯,并参与多种生理及病理过程.本文针对细胞分泌外泌体的动态过程,以DNA四面体为基础,结合细胞膜修饰技术和荧光成像技术,构建了一种易操作、高稳定性的细胞膜表面DNA四面体传感器,应用于不同细胞类型分泌外泌体的实时监测.DNA四面体传感器通过脚支链与疏水性胆固醇探针的杂交互补作用锚定于细胞膜表面,利用四跨膜蛋白CD63核酸适配体序列特异性捕获细胞膜表面释放的外泌体,通过监测细胞膜表面荧光的变化,实时测定外泌体的分泌情况.     

微流控技术在外泌体分离分析中的研究进展

外泌体是一类由细胞分泌的含有脂质、蛋白、核酸等多种物质的纳米级囊泡,主要参与细胞间的物质交换及信息传导,与多种疾病的发生发展密切相关。对外泌体进行深入研究,理解其生物学功能,对疾病诊断与治疗具有重要意义。由于外泌体尺寸较小且密度和体液接近,想要对复杂生物样本中的外泌体进行分离与分析十分困难。传统的外泌体分离方法如超速离心、超滤等大都需要借助大型仪器设备,且耗时长、操作复杂。因此迫切需要开发高效、便捷的外泌体分离检测手段。微流控技术因其微型化、高通量、可集成等特点,为外泌体的分离分析提供了一个新的平台。该文主要对近年来微流控技术在外泌体分离分析相关领域的研究进展进行了综述。重点从外泌体物理特性和生化特性两个角度出发,介绍了微流控芯片技术用于外泌体分离领域的主要原理、策略和方法。此外,还介绍了微流控技术与荧光、电化学传感、表面等离子体共振等多模态检测方法结合,实现外泌体一体化分析的新进展。最后,该文分析了目前微流控技术用于外泌体分离检测存在的挑战,并对其发展趋势和前景进行了展望。随着微流控外泌体分离分析装置的不断微型化、集成化、自动化,微流控芯片技术将在外泌体分离、生化检测、机制研究等方面将发挥越来越重要的作用。     

不同金属/适配体双功能复合磁性纳米材料的制备及其对外泌体的富集性能

外泌体作为一种细胞外囊泡,其内容物可以反映亲代细胞的重要信息,而自身也具有独特的结构,能够执行特征的生物学功能。基于外泌体的表面化学和生物学特征,制备了不同类型的金属/适配体(Apt)双功能复合磁性纳米材料,并将其应用于外泌体的富集纯化。将适配体和外泌体表面目标膜蛋白的特异性结合性能与以钛、锆为代表的金属氧化物和外泌体磷脂双层膜的特异性亲和作用结合,可极大地提高分离材料对外泌体的分离选择性和富集容量。分别以Fe₃O₄@Zr-MOFs、Fe₃O₄@Zr-Ti-MOFs和Fe₃O₄@TiO₂等金属有机框架(MOFs)/金属氧化物磁性纳米材料为基底,制备对应的双功能MOFs/金属氧化物-适配体复合磁性纳米材料Fe₃O₄@Zr-MOFs-Apt、Fe₃O₄@Zr-Ti-MOFs-Apt和Fe₃O₄@TiO₂-Apt,并进一步对不同材料的外泌体富集性能加以评价。以超速离心法提取的模型外泌体以及尿液为样品,对修饰相同质量适配体和不同含量金属氧化物的双功能材料的富集性能加以对比。将3种双功能磁性纳米材料应用于尿液外泌体的富集,得到的外泌体裂解后经质谱鉴定,分别得到233、343和832个外泌体蛋白。这一结果也表明双功能磁性纳米材料可以充分结合核酸适配体亲和的高选择性和金属氧化物的高富集容量优势,对于复杂生物样品中外泌体的快速、高效分离纯化具有潜在的应用价值,而针对材料制备和分离纯化方法的设计也为新型外泌体富集材料的设计提供了一条可行的新思路。     

外泌体:为高效药物投递策略提供天然的内源性纳米载体

在细胞通讯过程中,细胞分泌的大小在40~100 nm之间的外泌体具有负载"货物"并将其传递给靶细胞的能力。外泌体是细胞通讯重要的调节者,当疾病发生时,外泌体的组成会发生变化,在诊断和治疗方面均具有重要作用。外泌体作为药物的天然内源性载体具有独特优势,如其免疫原性低,在血液中的稳定性高,向细胞运输药物的效率高,和更强的增强渗透滞留效应(EPR)。目前,外泌体已经成功运载基因类药物、抗癌药物和抗炎症药物等其他类型药物。作为基因类药物载体,外泌体有助于同时提高转染效率和降低副作用。作为抗癌类和抗炎症类药物载体,外泌体可较好地保护药物免遭人体清除。本文按照外泌体负载"货物"的种类综述外泌体作为药物载体的最新进展,并简要讨论构建外泌体药物运输系统所需考虑的外泌体的来源、提纯方法、载药种类、载药方式以及载药系统的使用方式等关键因素对外泌体药物运输系统的影响,展望外泌体药物运输系统临床应用所面临的挑战和可能的解决途径。     

Proteomics comparison of exosomes from serum and plasma between ultracentrifugation and polymer‐based precipitation kit methods

Exosomes are vesicles with sizes ranging from 30 to 150 nm. The analysis and detection of blood exosomes offers an effective route for cancer diagnosis, prognosis assessment, and therapeutic evaluation of diseases. Due to the difference in separation procedure, collection method and the usage of anticoagulants, serum and plasma samples show diversity test results. In order to evaluate the isolation effect of exosomes in serum and plasma samples, two commonly used exosomal isolation methods, ultracentrifugation and polymer‐based precipitation kit, were used, respectively. And the isolation effects were evaluated by comparing the composition and abundant of proteins from isolated exosomes based on MS‐based proteomics analysis. The results showed that the plasma exosomes extracted by ultracentrifugation identified more exosome biomarkers, and the concentrations of these biomarkers were higher than others. And plasma exosomes could be a better sample for blood‐based proteomics research of exosomes. It would be more useful for future targeted biomarker discovery.     

Difference gel electrophoresis analysis of Ras-transformed fibroblast cell-derived exosomes

Exosomes are membrane vesicles of endocytic origin released by many cell types. The molecular composition of exosomes reflects the specialised functions of their original cells. For example, these vesicles can mediate communication through their ability to bind to target cells, facilitating processes such as vascular homeostasis and antigen presentation. Although the proteomes of exosomes from several cell types are known, exploration of exosomes from additional cell types may improve our understanding of their potential physiological roles. Here, we describe the isolation and characterisation of exosomes isolated from the culture medium of murine fibroblast NIH3T3 cells and Ras-transformed NIH3T3 cells. The vesicular nature and size (30-100 nm) of the purified fibroblast exosomes was confirmed by electron microscopy. 2-D difference gel electrophoresis (DIGE) was used to compare protein profiles of exosomes secreted from NIH3T3 cells and Ras-transformed NIH3T3 cells. LC-MS/MS sequencing identified proteins in 188 protein spots in the exosomes from the two cell lines, many of which have been previously identified in exosomes from other cell types. However, some proteins identified are novel for fibroblast exosomes, such as Serpin B6. Over 34 proteins, including milk fat globule EGF factor 8 (lactadherin), collagen alpha-1 (VI), 14-3-3 isoforms, guanine nucleotide-binding proteins (G proteins), the eukaryotic translation initiation factors elF-3 gamma and elF-5A accumulated (>2-fold) in exosomes upon Ras-induced oncogenic transformation. Significantly, the 10.4-fold increase in v-Ha-Ras p21 protein in exosomes derived from Ras-transformed NIH3T3 cells suggests that exosome secretion may be implicated in eradication of obsolete proteins.     

Exosome isolation and purification via hydrophobic interaction chromatography using a polyester,capillary‐channeled polymer fiber phase

Extracellular vesicles, including microvesicles and exosomes, are lipidic membrane‐derived vesicles that are secreted by most cell types. Exosomes, one class of these vesicles that are 30–100 nm in diameter, hold a great deal of promise in disease diagnostics, as they display the same protein biomarkers as their originating cell. For exosomes to become useful in disease diagnostics, and as burgeoning drug delivery platforms, they must be isolated efficiently and effectively without compromising their structure. Most current exosome isolation methods have practical problems including being too time‐consuming and labor intensive, destructive to the exosomes, or too costly for use in clinical settings. To this end, this study examines the use of poly(ethylene terephthalate) (PET) capillary‐channeled polymer (C‐CP) fibers in a hydrophobic interaction chromatography (HIC) protocol to isolate exosomes from diverse matrices of practical concern. Initial results demonstrate the ability to isolate extracellular vesicles enriched in exosomes with comparable yields and size distributions on a much faster time scale when compared to traditional isolation methods. As a demonstration of the potential analytical utility of the approach, extracellular vesicle recoveries from cell culture milieu and a mock urine matrix are presented. The potential for scalable separations covering submilliliter spin‐down columns to the preparative scale is anticipated.     

Advances in Exosomes‐Based Drug Delivery Systems

Exosomes, a subgroup of extracellular vesicles, are important mediators of long‐distance intercellular communication and are involved in a diverse range of biological processes such as the transport of lipids, proteins, and nucleic acids. Researchers, seeing the problems caused by the toxic effects and clearance of synthetic nanoparticles, consider exosomes as an interesting alternative to such nanoparticles in the specific and controlled transport of drugs. In recent years, there have been remarkable advances in the use of exosomes in cancer therapeutics or for treating neurological diseases, among other applications. The objective of this work is to analyze studies focused on exosomes used in drug delivery system, present and future applications in this field of research are discussed based on the results obtained.     

外泌体生物分析及其临床应用研究进展

外泌体是具有各种关键生物活性分子(如脂质、 蛋白质和核酸)的细胞外囊泡. 它们可以由所有类型的细胞分泌, 并分布在所有生物流体中, 如血液、 唾液、 汗水和尿液等. 更重要的是, 外泌体可以参与多种生理活动, 包括细胞间通讯、 哺乳动物的繁殖和免疫反应, 并在新陈代谢以及心血管疾病、 神经变性和癌症等疾病的病理进展中起重要作用, 这使其成为备受关注的天然非侵入性生物标记物, 并且被认为是可用于临床诊断和治疗的潜在工具。本文综述了用于检测外泌体的生物传感器的最新发展, 包括荧光、 电化学、 电化学发光、 表面增强拉曼光谱、 比色法和微流控技术等分析方法; 总结了在临床诊断和疾病治疗中外泌体的临床应用; 还讨论了外泌体检测所面临的挑战以及外泌体在临床诊断和疾病治疗等方面的应用潜力.     

Exosome-Hydrogel System in Bone Tissue Engineering: A Promising Therapeutic Strategy

Exosomes, as messengers of cell-to-cell communication, have many functional properties similar to those of their derived cells. Because they contain a large number of bioactive components that regulate recipient cell behavior, they are inanimate and do not require external maintenance or assistance. Various cell-derived exosomes are involved in many physiological processes of bone tissue repair. Hydrogels are widely used as scaffolding materials for bone tissue repair because their 3D network structure resembles the natural extracellular matrix. Moreover, their material properties and biological functions are adjustable. Exosomes can be delivered directly to the bone tissue damage site by hydrogel, and their duration of action in vivo can be prolonged by slow release. Therefore, the exosome-loaded hydrogel (Exo-Gel) system is a promising material for bone tissue engineering. In this study, the progress of the application of Exo-Gel in bone tissue repair and the improvement strategies, problems and research prospects of the current exosomes and hydrogels that have been applied to the Exo-Gel system for bone tissue repair are reviewed.     

A soluble pH-responsive host-guest-based nanosystem for homogeneous exosomes capture with high-efficiency

Exosomes offer ideal biomarkers for liquid biopsies. However, high-efficient capture of exosomes has been proven to be extreme challenging. Here, we report a soluble pH-responsive host-guest-based nanosystem (pH-HGN) for homogeneous isolation of exosomes around physiological pH. The pH-HGN consists of two specifically functionalized modules. First, a pH-responsive module, poly-dimethylaminoethyl methacrylate, provides homogeneous capture circumstances and sharp pH-triggered self-assembly separation in aqueous solution to improve capture efficiency and reduce nonspecific adsorption. Second, a host-guest module, poly-acrylamide azobenzene and β-cyclodextrin linked with exosomes-specific antibody, could act as the "cleavable bridge" to specific capture and subsequent rapid release of captured exosomes through host-guest interaction between β-cyclodextrin and AAAB moieties. The pH-HGN offered high capture efficiencies for exosomes from two different cell lines, which were 90.2% ± 0.28% and 87.0% ± 4.6% for H1299 and MCF-7 cell-derived exosomes, respectively. The purity of isolated exosomes was (1.49 ± 0.71) × 10¹¹ particles/µg, which was 4.1 times higher compared with the gold standard ultracentrifugation (UC) method. Furthermore, the isolated exosomes via the pH-HGN can preserve well integrity and biological activity. The developed pH-HGN was further successfully applied to differentiate lung cancer patients from healthy persons. These findings indicated that pH-HGN is a promising strategy in exosomes-based research and downstream applications.     

Multiple exosome RNA analysis methods for lung cancer diagnosis through integrated on-chip microfluidic system

Exosomes are now raising focus as a prospective biomarker for cancer diagnostics and prognosis owing to its unique bio-origin and composition. Exosomes take part in cellular communication and receptor mediation and transfer their cargos（e.g., proteins, m RNA and DNA）. Quantitative analysis of tumor-related nucleic acid mutations can be a potential method to cancer diagnosis and prognosis in early stages. Here we present an integrated microfluidic system for exosome on-chip isolation and lung can...