Research progress of extracellular vesicles

doi:10.3969/j.issn.2095-4344.2017.04.022

Abstract

Abstract:

Abstract
BACKGROUND: Extracellular vesicles (EVs) are a kind of subcellular component produced by paracine mechanism including exosomes, microparticles and microvesicles, which have become hotspots in recent years.
OBJECTIVE: To review the research status and progress of EVs, especially in the studies about definition, secreting mechanism, isolation and identification, biological characteristics and functions in diseases as well as in biomedical research.
METHODS: The first author retrieved PubMed and CNKI databases for relative articles published from July 2006 to August 2016. The keywords were “extracellular vesicles, exosome, microvesicle, microparticle” in English and Chinese, respectively.
RESULTS AND CONCLUSION: A total of 44 eligible literatures are enrolled. Almost all cells can secrete EVs, which contain a variety of metrocyte-derived bioactive molecules, such as lipids, proteins, mRNAs, microRNA, lncRNA, cicrRNA, and non-coding RNA. These bioactive molecules are encapsulated in EVs or binding with the membrane. EVs are described to be involved in inflammation, immunity, signal transduction, cell survival and apoptosis, angiogenesis, thrombogenesis, and autophagy, which are of great significance to the maintenance of homeostasis and disease progression. Special EVs may be used as new biomarkers for the diagnosis and prognosis of many diseases and serve as novel tools in the fields of antitumor therapy, regenerative medicine, immunoregulation and vaccination and drug delivery. But the molecular mechanisms regulating the secretion of EVs and the specific pathways activated upon EVs interaction with the target cell are not fully understood. Based on miRNA, lncRNA and circRNA are attracting researchers’ attention.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Tissue Engineering, Exosomes, Regeneration, Autophage, Drug Carriers

CLC Number:

R318

Wang Jin1, Chen Jian-ying2 . Research progress of extracellular vesicles[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(4): 621-626.

Figures/Tables 2

胞活化、氧化应激、细胞癌性转化、放射损伤、细胞死亡和(或)凋亡等[16]，如骨髓间充质干细胞经低氧或低营养诱导后会分泌释放细胞外囊泡[17]。细胞外囊泡形成、释放的分子机制不完全清楚，可能与细胞膜重构和细胞骨架改变有关。既往蛋白质组学、基因芯片分析及RT-PCR等显示细胞外囊泡选择性地将母细胞来源的多种生物活性物质包装进其双分子层膜结构中或携带于膜表面，如脂质(如富含胆固醇和鞘磷)、细胞因子、趋化因子、生长因子、特异性及非特异性蛋白、DNA、mRNA、microRNA、lncRNA、circRNA等[14，18-21]。外泌体：分子直径30-100 nm，也有很多学者认为在150 nm范围内的，来源于细胞质膜内陷的初级核内体的微粒都属于外泌体，这类囊泡在细胞内部形成，与一般的出芽方式不同。其形成释放过程是，母细胞释放颗粒物质到质膜内陷形成的核内体腔内囊泡中，含有腔内囊泡的核内体成为次级核内体，也称作多囊泡胞内体或多泡小体。随后，多泡小体离开溶酶体途径而与母细胞胞膜特定部位融合，之后胞内体中的微小囊泡朝向内体囊腔的内部形成芽泡，继而母细胞将这种芽泡以外泌的形式释放到细胞外。由于外泌体特殊的分泌方式，其所含蛋白组分有别于其他细胞外囊泡，不含有内质网内的蛋白质，而高表达与主要组织相容性复合体以及整合素等多种蛋白质相互作用的四分子交联体超蛋白家族，如CD9，CD81，CD63以及热休克蛋白，如热休克蛋白60，70，90和部分细胞内源性蛋白如Alix，Tsg101等，低表达磷脂酰丝氨酸。微囊泡：分子直径100-1 000 nm，也有文献称之为脱落囊泡或Ectosomes。形成过程相对简单，是直接通过出芽方式从母细胞膜表面脱落产生，大小不均一，高表达磷脂酰丝氨酸，没有特定的表面分子标记物，但和外泌体一样表达母细胞来源的表面标记物[22]。如EMV表达CD31，PMV表达CD42b，白细胞来源的表达CD45，网织红细胞来源的表达转铁蛋白受体分子，抗原呈递细胞来源的富含MHC-Ⅰ，Ⅱ。微囊泡中也含有如金属蛋白酶等外泌体中不包含的一些物质。凋亡小体：是细胞程序性死亡或凋亡晚期释放的微粒，文章不赘述，这类囊泡颗粒较大，分子直径为500- 4 000 nm，具有Annexin V高亲和力，富含死细胞(凋亡细胞)碎片的浓缩DNA，能将这些DNA运载入巨噬细胞细胞核中。 2.3 细胞外囊泡分离提取和鉴定方法目前，学界普遍认同差速离心法是提取细胞外囊泡的标准方法[23]。先低速离心去除死亡的细胞和大的细胞碎片，再高速离心去除可溶性蛋白质、蛋白质聚集体及其他与细胞外囊泡共沉淀的杂质，即可分离得到微囊泡和外泌体。对细胞外囊泡纯度要求较高时可采用密度梯度离心或免疫磁珠分选的方法来纯化细胞外囊泡。前者是在超速离心力作用下，使蔗糖溶液形成从低到高连续分布的密度阶层，是一种区带分离法。免疫磁珠分选技术则是利用抗原抗体特异性结合的原理，用包被抗标记物抗体的磁珠与细胞外囊泡孵育后结合，这样就能将特定的细胞外囊泡分选出来。此外，分离提取细胞外囊泡的方法还有诸如微孔过滤技术、微流控技术、高效液相色谱法以及细胞外囊泡提取试剂盒。随着研究的深入，目前对细胞外囊泡也进行蛋白、DNA/RNA提取，有学者使用传统方法提取，也有研究者用BioVision等公司的提取试剂盒，总的来说各种技术各有利弊。关于细胞外囊泡的鉴定，已有许多技术能表征以及分析纳米颗粒和纳米囊泡。其中包括：蛋白免疫印迹、流式细胞学、酶联免疫吸附分析、动态光散射、透射电镜检查法、纳米颗粒跟踪分析技术。蛋白免疫印迹和酶联免疫吸附分析技术是对细胞外囊泡中蛋白进行分析，流式细胞学技术能分析细胞外囊泡携带的细胞表面抗原标记物；透射电镜检查法技术可以分析细胞外囊泡分子直径和形态结构；纳米颗粒跟踪分析技术可实时地对低浓度的细胞外囊泡悬浮液中50-1 000 nm直径范围内特定的细胞外囊泡进行分子波峰及浓度测定。 2.4 细胞外囊泡生物学功能 2.4.1 细胞外囊泡所携带信息物质目前Vesiclepedia中记录了来自33种不同种类的538份研究统计分析出细胞外囊泡中含有92 897种蛋白，27 642 种mRNAs， 4 934种 miRNAs和584种脂质等(数据是在2015年9月前收录)[24]。蛋白质谱分析发现这些蛋白包括血管内皮生长因子、碱性成纤维细胞生长因子、血小板衍生生长因子、转化生长因子β、MAPK通路信号分子、RHO通路信号分子、Tie-2/TEK及细胞黏附分子等。这些蛋白质分子部分是酶类，酶催化具有高效性，能够放大其效应，相比RNA，进入内皮细胞的蛋白质能够即时迅速发挥作用，在维持血管新生以及维持血管功能及修复损伤组织等方面起主要作用。Ratajczak等[25]证实小鼠胚胎干细胞来源的MVs能把蛋白质和mRNA转运到造血祖细胞中，并使其重新编程，这一作用经RNase处理微粒后不复存在，表明有微粒转运的mRNA不仅能稳定存在于靶细胞，还能在靶细胞内被翻译成相应蛋白。近年来有研究者报道在这类囊泡中发现lncRNA、circRNA等noncodingRNA及gDNA[20-21，26]。microRNA (miRNA)是一类长约22 nt且高度保守的单链小分子RNA，本身不编码蛋白质但能够在转录后水平调控基因的表达。且一个miRNA能够调控多个mRNA，而一个mRNA能同时被多个miRNA调控，目前已知miRNA的功能有调控细胞的增殖、分化和代谢，并与心血管疾病、肿瘤、白血病等疾病的发生、发展有着密切联系。lncRNA即长链非编码RNA，是一类本身不编码蛋白、转录本长度超过200 nt的长链非编码RNA，能干扰mRNA的剪切，他在表观遗传调控、转录调控以及转录后调控等多层面调控基因的表达。circRNA即环状RNA，是一类特殊的非编码RNA分子，富含miRNA结合位点，在细胞中起到miRNA海绵的作用，进而解除miRNA对靶基因的抑制作用，升高靶基因的表达水平。Collino等[27]通过miRNA芯片对比骨髓MSC和肝脏MSC与两者分泌的微粒发现一些miRNA同时存在于微粒和来源母细胞中，一些miRNA只存在于母细胞中，一些miRNA则只存在微粒中。Chen等[19]的一项人脐带间充质干细胞来源的微囊泡成血管相关蛋白分析中关于细胞外囊泡中蛋白质成分分析也有类似发现，目前这种现象发生的机制仍不明确有待进一步研究。 2.4.2 细胞外囊泡参与细胞间物质、信息传递在生理、病理条件下，细胞外囊泡将其携带生物信息运输到周边靶细胞或经血液循环及体液运输而被远处组织细胞摄取，进而对靶细胞遗传组进行重新编排，使靶细胞获得新的功能或失去某功能甚至死亡[28]。一项研究表明成纤维细胞来源的、携带有miR-195的细胞外囊泡能够在胆管癌大鼠体内富集，降低肿瘤大小，改善治疗鼠的存活[29]。细胞外囊泡作用于靶细胞参与细胞间信息传递主要有3种方式：第1种，通过其携带的特异性表面分子配体与靶细胞上相应受体位点结合；第2种，直接与靶细胞质膜融合，释放其内容物到靶细胞胞质中。第3种，通过内陷以类似胞吞作用的机制将信息直接传递给靶细胞(图3)。 2.5 细胞外囊泡参与疾病的发生发展中枢神经细胞来源的细胞外囊泡可以进入血液循环并在外周循环中发挥其促增殖、分化及抗凋亡的作用，在阿尔茨海默病和帕金森病等退行性疾病及其他中枢神经系统疾病中发挥着重要作用。内皮细胞来源细胞外囊泡本身与机体高凝倾向和小血管炎症等微循环障碍疾病有密切关系[30]。T细胞来源的外泌体可通过磷脂酰丝氨酸识别单核细胞膜表面的受体，诱导胆固醇堆积，进而促进动脉粥样硬化形成[31]。类风湿性关节炎患者关节滑液来源的纤维母细胞分泌的细胞外囊泡携带的TNF-α结合于T淋巴细胞后抵抗机体活化诱导的细胞死亡，从而加重关节炎症状[32]。细胞外囊泡也参与体内自噬过程，有研究指出，经烟雾提取物诱导人气道上皮细胞后收集到的HBEC-细胞外囊泡中miR-210表达量上调，后者作"

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