卵泡液中细胞外囊泡及其携带的microRNA对卵泡发育的作用

细胞外囊泡(Extracellular vesicles,EVs)是指细胞分泌的双层膜转运囊泡。EVs能从细胞中摄取大分子物质,并将其转移至受体细胞。在这些大分子物质中,研究最多的就是microRNA (miRNA)。miRNA是一种参与基因表达调控的非编码RNA,已证实在哺乳动物卵泡液EVs中有不同的非编码RNA存在,EVs携带miRNA可以作为自分泌和旁分泌的替代机制,影响卵泡发育。文中系统介绍了EVs的种类、特征和分离鉴定方法,重点综述了EVs及携带的miRNA对卵泡发育的作用,包括早期卵泡发育、卵母细胞成熟、卵泡优势化以及对颗粒细胞功能的影响。同时对卵泡液中EVs及其携带的miRNA的未来研究进行了展望,为卵泡液中EVs及携带的miRNA功能的研究及应用提供了思路和方向。     

细胞外囊泡在肾脏病治疗中的研究进展

细胞外囊泡(extracellular vesicles, EVs)是细胞分泌的由脂质双分子层包绕形成的膜性囊泡,其内包含丰富的生物学信息,参与多种生理和病理过程。近年来,EVs在疾病治疗中也展现出了巨大应用前景,一方面来源于干细胞的EVs可以直接发挥治疗作用,另一方面EVs还可以作为一种天然的药物递送载体,为疾病的治疗提供新的给药策略。本文聚焦EVs在肾脏病治疗中的应用和研究进展。     

正常与氧糖剥夺再复氧培养的星形胶质细胞来源外泌体miRNA测序分析

本研究通过高通量测序技术,分析正常培养和氧糖剥夺再复氧(oxygen and glucose deprivation/reoxygenation,OGD/R)培养星形胶质细胞来源外泌体的差异微小RNA(microRNA,miRNA)。采用超速离心法提取正常组和OGD/R组星形胶质细胞培养基上清的外泌体,透射电镜观察到提取的外泌体呈典型囊泡状,包膜完整,含有低电子密度的物质;纳米颗粒追踪技术(NTA)检测到星形胶质细胞外泌体大小为100.5±31.1 nm,占比为 96.8%;免疫印迹检测显示,提取物中有外泌体标志性蛋白肿瘤易感蛋白(tumour-susceptibility protein, TSG101)、热休克蛋白60 (heat shock proteins 60, Hsp60)、ALG-2相互作用蛋白X(ALG-2-interacting protein X, ALIX)的表达。与正常组相比,OGD/R组共有41个miRNA发生显著改变,其中20个miRNA显著升高,21个miRNA显著降低(P<0.05)。基因本体功能(GO)分析显示,差异靶基因主要参与蛋白质糖基化、脂质代谢过程、磷酸化作用、高尔基体、内质网、内吞体、细胞质囊泡和细胞突起等生物学过程;京都基因与基因组百科全书(KEGG)通路分析发现,差异靶基因主要与丁酸代谢、β-丙氨酸代谢、脂肪酸降解、线粒体自噬和P53信号通路等代谢途径和信号通路有关。通过对正常组和OGD/R组的星形胶质细胞来源的外泌体miRNA测序并进一步施行靶基因功能富集分析,为后续研究星形胶质细胞外泌体对氧糖剥夺再灌注神经元发挥的保护作用的具体机制提供了一定的研究基础。     

胞外囊泡表面糖缀合物研究进展

胞外囊泡(extracellular vesicles,EVs)是一类由细胞分泌到胞外的能够被受体细胞摄取的膜性囊泡小体,直径在20~1 000 nm.近年来,越来越多的研究者发现胞外囊泡在疾病诊断、预后评估以及药物递送等方面具有重要的生物学作用.胞外囊泡可以直接参与细胞间信息的传递以及物质的运输,其携带的核酸(m RNA,micro RNA和lnc RNA)和蛋白质可以影响受体细胞的生理状态.大量研究表明,胞外囊泡是被糖基化修饰的,胞外囊泡表面覆盖了大量的聚糖以及糖结合蛋白,而已知聚糖类物质在调控细胞黏附、细胞-细胞之间的信息传递、细胞和细胞外基质相互作用、免疫调节和肿瘤转移等方面发挥重要的作用.本文综述了近年来细胞外囊泡表面糖缀合物修饰的前沿研究,以期更好地理解聚糖在胞外囊泡的合成、释放以及运输过程及其生物学功能中的作用.     

细胞外囊泡在非酒精脂肪肝发病机制及诊断治疗中的作用

非酒精性脂肪性肝病(nonalcoholic fatty liver disease, NAFLD)是21世纪全球最重要的公共健康问题之一,也是我国愈来愈重要的慢性肝病问题。细胞间通讯在NAFLD病理进程中发挥重要作用。细胞外囊泡(extracellular vesicles, EVs)是近年来备受关注的细胞间通讯方式。EVs携带脂质、蛋白质、DNA、mRNA以及非编码RNA等作为信号分子在细胞间的物质和信息交流中起重要作用,参与了多个生理病理过程。目前细胞外囊泡在非酒精脂肪肝发病机制及诊断治疗中的作用方面的研究非常有限,但初步研究显示, EVs在NAFLD病程发展中发挥重要作用。因此,该文重点关注EVs参与NAFLD病程机制研究,并对其在NAFLD防治中的潜在诊疗价值作简要综述。     

一种细胞外囊泡的流式检测方案的探索与建立

该研究旨在探索细胞外囊泡(extracellular vesicles,EVs)分析的标准化流程,建立一种高效的EVs检测方法,为EVs功能及临床转化研究提供技术支撑。实验采用经典的超速离心法分离EVs,借助已知直径的聚苯乙烯微球设定并优化流式检测EVs的参数,联合应用散色光信号及荧光信号对EVs进行双参数分析鉴定,最后通过电镜观察及蛋白质免疫印迹(Western blot,WB)对鉴定结果进行验证。结果显示,参照纳米微球设定的条件可清楚地将100 nm颗粒信号与噪音信号分离,且微球稀释的梯度和检测到的浓度之间呈线性相关,可以对EVs进行定量分析;荧光染色结果显示,EVs呈CFSE(5,6-Carboxyfluorescein diacetate,succinimidyl ester,蛋白结合染料)及FM1-43(fixable analog of FM?1-43 membrane stain,亲脂性染料)双阳性,占比约3%;电镜验证其形态及直径与预期相一致。该研究通过优化流式方案及双荧光参数分析鉴定探索了EVs流式检测的标准化流程,建立了高效、准确的EVs流式检测方法。     

细胞外囊泡在肥胖引起的胰岛素抵抗中的作用

肥胖是世界范围内一个重要的公共健康问题,通常与代谢紊乱的发展,尤其是胰岛素抵抗(insulin resistance, IR)息息相关。细胞外囊泡(extracellular vesicles, EVs)包含外泌体、微泡(microvesicles,MVs)和凋亡小体,EVs一般指外泌体和微泡,能够携带功能蛋白、核酸和脂质,作为细胞间通信系统的一部分,由于能在各种病理状态下发挥生物学功能,现已广泛引起人们的关注。肥胖引起的IR中脂肪组织释放的EVs是细胞通信中不可忽略的重要调控者。然而,目前还不确定EVs能否可作为可靠的用于诊断早期发现肥胖或糖尿病中IR的标志物。本文介绍了EVs的形成机制,并回顾了脂肪组织中IR的主要调节方式,总结了近些年关于EVs在肥胖引起的IR中的作用,以及EVs与糖尿病、代谢综合征等疾病的相关性。     

RNA修饰类型及调控蛋白

RNA修饰是指发生在RNA上的各种修饰形式。自然界中的RNA修饰广泛存在于A、U、C、G四类核苷酸上,此外,极少的RNA修饰发生在次黄嘌呤核苷(I)上。目前已经在古细菌、细菌、病毒和真核生物中发现超过140种的RNA转录后修饰形式。在各种类型的RNA修饰中,甲基化修饰占到了三分之二,这些修饰广泛存在于各种RNA类型中,包括信使RNA(mRNA)、转运RNA(tRNA)、核糖体RNA(rRNA)、核内小RNA(snRNA)、核仁小RNA(sno RNA)、微小RNA(miRNA)、小干扰RNA(siRNA)、piwi蛋白相互作用的RNA(piRNA)和长非编码RNA(lncRNA)等。现介绍主要的RNA修饰类型,并对其调控蛋白进行归纳总结。     

胞外和胞内菌胞外囊泡的功能及应用

细胞外囊泡(Extracellular Vesicles, EVs)是从细胞膜上脱落或者分泌的双层膜结构的囊泡状小体。真核生物、细菌、古细菌和支原体等具有细胞结构的生物均能够释放EVs。细菌分泌的EVs含有DNA、RNA及蛋白质等多种成分,其在细菌毒力保持、免疫逃逸、细菌间物质运输、宿主细胞免疫调节、宿主转录基因调节、耐药性等方面发挥巨大作用。细菌可以分为胞内菌(Intracellular bacteria)和胞外菌(Extracellular bacteria),二者EVs在释放机制、生理学功能和临床应用等方面存在着区别与联系。就胞内菌和胞外菌的囊泡分泌机制、功能及应用作一阐述。     

运动调控胞外囊泡生物发生的研究进展

胞外囊泡(extracellular vesicles, EVs)是一类由细胞释放的各种具有膜性结构的囊泡的统称,其能被大多数细胞所分泌,通过携带核酸、蛋白质、脂质等特定的生物活性分子到达靶细胞实现细胞间信息交流。目前EV的生物发生调节已成为疾病防治的关键靶点,受到广泛关注。运动能够促进EV的释放,调节其内容物构成,上述作用甚至被认为是机体对运动产生适应的标志。运动时信号通路(如Ca²⁺、mTOR、STAT3等)的激活,细胞微环境(如pH和氧浓度等)乃至EV生物发生相关蛋白质翻译后修饰的变化都会影响EV的形成、内容物分选及囊泡运输等过程。该综述总结了EV的生物发生过程、运动对EV生物发生的影响,并根据现有研究,从信号通路、细胞微环境及蛋白质翻译后修饰的角度分析运动调节EV生物发生的可能机制。     

Proteomic Analysis of Extracellular Vesicles for Cancer Diagnostics

Extracellular vesicles (EVs) including exosomes and microvesicles are lipid bilayer‐encapsulated nanoparticles released by cells, ranging from 40 nm to several microns in diameter. Biological cargoes including proteins, RNAs, and DNAs can be ferried by EVs to neighboring and distant cells via biofluids, serving as a means of cell‐to‐cell communication under normal and pathological conditions, especially cancers. On the other hand, EVs have been investigated as a novel “information capsule” for early disease detection and monitoring via liquid biopsy. This review summarizes current advancements in EV subtype characterization, cancer EV capture, proteomic analysis technologies, as well as possible EV‐based multiomics for cancer diagnostics.     

Extracellular vesicles of blood plasma: content,origin, and properties

Extracellular vesicles (EVs) are bilayer membrane fragments that are released by different cell types upon activation or death. The most well studied EVs are those of blood plasma. Two types of EVs are usually distinguished: exosomes (formed by the membranes of intracellular compartments, 50–100 nm in diameter) and ectosomes (also called microparticles or microvesicles, formed from plasma membrane, 100–1000 nm in diameter). The real picture is much more complicated and is still poorly understood. EVs are enriched by various proteins, mRNA and miRNA, and the EV lipid and protein composition can substantially differ from that of the parental cells, from which EV originate. The blood concentration of EVs greatly increases in many diseases and conditions. EVs have a wide spectrum of biological activities, from pro-coagulant to immunomodulating ones. This activity can be physiologically important and is believed to be absolutely important pathophysiologically. In recent studies, EVs are considered to be important not only as objects of basic research, but also as potential biomarkers, drug candidates, drug carriers, or therapeutic targets.     

肠道菌群来源细胞外囊泡在肝脏疾病中的作用研究进展

细胞外囊泡(extracellular vesicles, EVs)是一类具有脂质双分子层的膜性囊泡,可以被各种类型细胞分泌,是生物体通信的重要介质,参与原核生物和真核生物细胞之间的信号传输。在肠道微生态中,微生物-宿主的双向通信通常不需要细胞直接接触,微生物群来源EVs是这种“跨界”对话的关键参与者。肠-肝轴是连接肠道微生物与肝脏的桥梁,参与包含酒精性脂肪性肝病在内的多种肝脏疾病的发生与发展,近年研究发现肠道菌群来源的EVs在肝脏疾病的进程中具有重要的调控作用。本文概述了肠道菌群来源EVs的研究进展,特别是EVs的产生机制、包裹的内容物、在细菌-宿主互作以及在肝脏疾病中的作用。     

葡萄球菌细胞外囊泡研究进展

细胞外囊泡(extracellular vesicles,EV)是由脂质双分子层包裹着蛋白质、核酸等生物分子组成的天然纳米结构颗粒。EV作为细胞间无细胞通讯的一种方式,通过传递包括遗传信息在内的大量生物分子来影响细胞间的通讯。此外,EV还参与多种生物学功能的调控,如免疫调节、细胞间竞争、水平基因转移和致病性等。革兰氏阳性细菌分泌的EV携带多种化合物,这些化合物在细菌竞争、生存、入侵、抗生素耐药和感染方面发挥多样化作用。目前对于细菌EV的研究主要集中在革兰氏阴性细菌中,对革兰氏阳性细菌EV的研究报道较少,其中对葡萄球菌EV的报道主要是金黄色葡萄球菌和表皮葡萄球菌。这篇综述介绍了葡萄球菌EV的化学成分组成、功能和分泌的影响因素及临床应用。     

哺乳动物精浆胞外囊泡及各组分的功能

精浆胞外囊泡是一种存在于精浆的膜性囊泡,按分泌器官分为附睾小体和前列腺小体。囊泡可与精子细胞膜发生融合,通过传递内容物或介导信号通路进而调节精子功能。它含有多种活性物质,其中蛋白质组分可影响精子活力以及顶体反应,并有清除损伤精子和促进细胞粘附的作用;脂质组分具有调节靶细胞质膜稳定性的作用;核酸组分主要参与免疫反应、跨代遗传及男性不育;离子则是多种酶的辅助因子,在调节酶活性和精浆微环境中发挥重要作用。不同组分对精子功能的影响不尽相同,本文将对此方面的研究进展进行详尽的综述,以期为该领域相关研究人员提供一定的参考。     

Interaction of the 140/130/110 kDa rhoptry protein complex of Plasmodium falciparum with the erythrocyte membrane and liposomes

During Plasmodium falciparum merozoite invasion into human and mouse erythrocytes, a 110-kDa rhoptry protein is secreted from the organelle into the erythrocyte membrane. In the present study our interest was to examine the interaction of rhoptry proteins of P. falciparum with the erythrocyte membrane. It was observed that the complex of rhoptry proteins of 140/130/110 kDa bind directly to a trypsin sensitive site on intact mouse erythrocytes, and not human, saimiri, or other erythrocytes. However, when erythrocytes were disrupted by hypotonic lysis, rhoptry proteins of 140/130/110 kDa were found to bind to membranes and inside-out vesicles prepared from human, mouse, saimiri, rhesus, rat, and rabbit erythrocytes. A binding site on the cytoplasmic face of the erythrocyte membrane suggests that the rhoptry proteins may be translocated across the lipid bilayer during merozoite invasion. Furthermore, pretreatment of human erythrocytes with a specific peptide derived from MSA-1, the major P. falciparum merozoite surface antigen of MW 190,000-200,000, induced binding of the 140/130/110-kDa complex. The rhoptry proteins bound equally to normal human erythrocytes and erythrocytes treated with neuraminidase, trypsin, and chymotrypsin indicating the binding site was independent of glycophorin and other major surface proteins. The rhoptry protein complex also bound specifically to liposomes prepared from different types of phospholipids. Liposomes containing PE effectively block binding of the rhoptry proteins to mouse cells, suggesting that there are two binding sites on the mouse membrane for the 140/130/110-kDa complex, one protein and a second, possibly lipid in nature. The results of this study suggest that the 140/130/110 kDa protein complex may interact directly with sites in the lipid bilayer of the erythrocyte membrane.     

Combination of size-exclusion chromatography and ion exchange adsorption for improving the proteomic analysis of plasma-derived extracellular vesicles

Extracellular vesicles (EVs) are lipid membrane vesicles released by live cells that carry a variety of biomolecules, including nucleic acids, lipids, and proteins. Recently, proteins in plasma-derived EVs have emerged as novel biomarkers with essential functions in the diagnosis and prognosis of human diseases. However, the current methods of isolating EVs from plasma often lead to coisolated impurities in biological fluids. Therefore, before performing any research protocol, the process of extracting EVs from plasma for proteomic analysis must be optimized. In this study, two EV isolation strategies, size exclusion chromatography (SEC) and SEC combined with ion exchange adsorption (SEC + IEA), were compared in terms of the purity and quantity of protein in EVs. Our results demonstrated that, compared to single-step SEC, SEC combined with IEA could produce plasma-derived EVs with a higher purity by decreasing the abundance of lipoprotein. Additionally, with MS analysis, we demonstrated that the combination approach maintained the stability and improved the purity of EVs in many plasma samples. Furthermore, by combining SEC with IEA, more cancer-associated proteins were detected in the plasma of various cancer samples.     

Lipid/myelin basic protein multilayers. A model for the cytoplasmic space in central nervous system myelin

A multilayered complex forms when a solution of myelin basic protein is added to single-bilayer vesicles formed by sonicating myelin lipids. Vesicles and multilayers have been studied by electron microscopy, biochemical analysis, and X-ray diffraction. Freeze-fracture electron microscopy shows well-separated vesicles before myelin basic protein is added, but afterward there are aggregated, possibly multilayered, vesicles and extensive planar multilayers. The vesicles aggregate and fuse within seconds after the protein is added, and the multilayers form within minutes. No intra-bilayer particles are seen, with or without the protein. Some myelin basic protein, but no lipid, remains in the supernatant after the protein is added and the complex sedimented for X-ray diffraction. A rather variable proportion of the protein is bound. X-ray diffraction patterns show that the vesicles are stable in the absence of myelin basic protein, even under high g-forces. After the protein is added, however, lipid/myelin basic protein multilayers predominate over single-bilayer vesicles. The protein is in every space between lipid bilayers. Thus the vesicles are torn open by strong interaction with myelin basic protein. The inter-bilayer spaces in the multilayers are comparable to the cytoplasmic spaces in central nervous system myelins . The diffraction indicates the same lipid bilayer thickness in vesicles and multilayers, to within 1 A. By comparing electron-density profiles of vesicles and multilayers, most of the myelin basic protein is located in the inter-bilayer space while up to one-third may be inserted between lipid headgroups. When cytochrome c is added in place of myelin basic protein, multilayers also form. In this case the protein is located entirely outside the unchanged bilayer. Comparison of the various profiles emphasizes the close and extensive apposition of myelin basic protein to the lipid bilayer. Numerous bonds may form between myelin basic protein and lipids. Cholesterol may enhance binding by opening gaps between diacyl-lipid headgroups.     

Large extracellular vesicles: Size matters in tumor progression

Extracellular Vesicles (EVs) represent a heterogeneous population of particles naturally released from all cells, delimited by a lipid bilayer and able to horizontally transfer their cargos to recipient cells. These features imply the growing interest on EVs in cancer biology as biomarkers and therapeutic targets. In this review, we will highlight the specific process related to biogenesis and release of large EVs (L-EVs) derived from the plasma membrane (PM) compared to the small and well described exosomes, generated through the classical endosome-multivesicular body (MVB) pathway. The control of PM rigidity by cells depends on lipid/protein composition, cytoskeleton dynamics, cytoplasmic viscosity, ions balance, metabolic reprogramming and specific intracellular signaling pathways, all critical determinants of L-EVs biogenesis. We will focus in details on a specific class of L-EVs, named Large Oncosomes (LO), exclusively shed by cancer cells and with a size ranging from 1 μm up to 10 μm. We will examine LO specific cargos, either proteins or nucleic acids (i.e. mRNA, microRNAs, single/double-stranded DNA), as well as their functional role in cancer development and progression, also discussing the mechanisms of L-EVs internalization by recipient cells. Overall we will highlight the potential of LO as specific diagnostic/prognostic cancer biomarkers discussing the associated challenges.