干细胞来源的胞外囊泡对T细胞的免疫调控作用

胞外囊泡（EVs）是细胞旁分泌产生的一种亚细胞成分,实质上是一组纳米级颗粒。它是双层膜结合型囊泡,内含蛋白质、核酸等活性成分。EVs在细胞间通过转移携带的信号分子而获得重要的地位。目前关于EVs在体外和体内的研究中对T细胞的调控能力引起了人们广泛的兴趣。在大多数研究中干细胞被报道能够抑制T细胞的增殖、活化和分化,在极少数研究中也发现干细胞具有增强T细胞免疫反应的作用。事实上所有的细胞类型均能释放EVs,包括干/祖/前体细胞。EVs被认为是细胞间交流的一种新机制,具有与干/祖细胞等亲代细胞相似的免疫调控作用。本综述是概述干/祖细胞来源的EVs对T细胞调控作用及可能的机制。     

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。     

烟曲霉的细胞外囊泡蛋白质及RNA分析

目的 分析烟曲霉的细胞外囊泡(extracellular vesicles, EVs)中的重要成分,以进一步明确曲霉致病机制。方法 通过离心法分离烟曲霉的囊泡,用电镜观察形态。采用马尔文纳米颗粒跟踪分析仪分析溶液中EVs的大小分布。通过质谱仪对囊泡内处理后的肽段进行分析。二级质谱数据使用Maxquant (v1.5.2.8)进行检索。检测RNA分布,通过数据库分析烟曲霉EVs中miRNA可能参与的一些通路。结果 电镜下烟曲霉的EVs可见明显的双层脂质结构。NTA发现烟曲霉分析的EVs大小主要集中在130 nm左右。EVs蛋白中不稳定蛋白为9种(15%),其余为稳定蛋白,而等电点(isoelectric point, PI)>7的为6种蛋白。EVs中大部分是胞浆蛋白,其余比较多的是细胞外分泌蛋白,但仍有25%的蛋白不能定位。通过跨膜蛋白预测(transmembrane prediction, TMPred)和糖基磷脂酰肌醇(glycosylphosphatidylinositol, GPI)预测,有5种蛋白存在于双层脂质膜上的蛋白。检测到RNA中rRNA和tRNA分别占59.7%和...     

细菌胞外囊泡的研究进展

胞外囊泡(extracellular vesicles,EVs)是自然界中细胞生命活动的产物，是一种包裹核酸、蛋白、脂类等分子的纳米级磷脂双分子层颗粒。近年来，越来越多的研究证实细菌可以分泌EVs作为抗生素和噬菌体的“诱饵”，从而发挥防御功能；此外，EVs还在传递毒力因子、细胞间通讯、介导基因水平转移、营养和电子传递、促进生物膜的形成中发挥重要作用。因此，EVs对生物个体和群体都具有十分重要的作用。本文综述了细菌EVs形成机制、提取及鉴定方法、影响EVs分泌的因素等，重点总结了EVs的生物学功能以及在环境科学领域的研究进展，为EVs的进一步研究提供参考。     

益生菌胞外囊泡与疾病的相关研究进展

益生菌是对宿主健康有益的活的微生物,具有广泛的促健康作用。它可以缓解和改善肠道相关炎症性疾病的症状,降低血清胆固醇,调节肠道菌群平衡,对恶性肿瘤的治疗和预防有重要作用。胞外囊泡(extracellular vesicles, EVs)是革兰阴性菌及少数革兰阳性菌释放的含有多种生物活性物质的一种纳米级囊泡结构。EVs通过携带生物信息分子在细菌与细菌或细菌与宿主之间的交流中起着非常重要的作用。虽然近年来研究者们对革兰阳性和阴性病原体来源的EVs的研究越来越多,但对益生菌产生的EVs的研究却很少。本文综述了近年来益生菌释放的EVs在治疗相关疾病中的研究进展,并对未来的发展形势进行分析。希望本文能围绕益生菌EVs这一新兴领域的最新进展,寻找基于EVs的相关疾病的诊断工具和有效治疗方法。     

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。     

真菌细胞外囊泡的研究进展CSCD

细胞外囊泡(extracellular vesicles,EVs)是一种细胞分泌的膜性囊泡。研究表明EVs在细胞生命的许多方面起着重要作用,包括细胞间的通讯、发病机制和癌症进展[1]。然而,EVs在微生物学中的作用却研究较少,EVs在微生物中最先是在革兰氏阴性菌中发现的[2],后续的研究表明它们在革兰氏阳性菌中也普遍存在[3],此后也有研究发现其在病毒致病过程中的作用和机制。目前,关于EVs与真菌感染关系的研究甚少,本综述将介绍EVs在真菌中的相关研究进展。     

基于虚拟仪器技术的血管壁动态信息无创检测与辅助诊断系统

为了在体外无损地实现对血管壁动态信息、心电和心音信号等医学信息多参数的综合同步检测以及分析和处理,利用虚拟仪器技术设计了血管壁动态信息多参数的无创检测辅助诊断系统．该系统硬件平台由信号输入模块、信号调理模块以及采集卡等三大模块组成。软件系统由开发虚拟仪器的流行软件LabVIEW编写,实现了数据的采集、实时显示、分析处理和存储等。初步的临床检测结果证实了本无创检测系统的可行性和临床应用的前景,为功能性无创辅助诊断与血管壁弹性（硬化）程度有关的血管疾病提供了一种新的方法。     

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。     

DNA芯片的制作原理及其应用

综述了DNA芯片制作原理和杂交信号检测方法及发展趋势,对DNA芯片在研究基因结构和基因表达等方面的应用进行了分析。     

Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry

BACKGROUND: We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres. METHODS: A fluoresceinated oligonucleotide reporter sequence is added to a "capture" probe by OLA. Capture probes are designed to hybridize both to genomic "targets" amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called "ZipCodes". The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNP genotype. RESULTS: Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere-based SNP analysis agreed with genotyping by sequencing in all cases. CONCLUSIONS: Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs.     

Flow cytometry-based biosensor for detection of multivalent proteins

Microsphere-based flow cytometric detection of cholera toxin (CT) through distance-dependent fluorescence resonant energy transfer (FRET) has been developed. Simultaneous double-fluorescence changes induced by multivalent interactions between CT and fluorophore (both fluorescence donor and acceptor)-labeled ganglioside GM1 on a biomimetic membrane surface (supported bilayers of phospholipids) can be measured by a commercial flow cytometer, providing a convenient and sensitive detection method for CT. The flow cytometry-based biosensor is capable of detecting less than 10 pM CT within 30 min. The signal generation strategy coupled with flow cytometry also provides a convenient method for kinetic studies of multivalent interactions. The surface density and the ratio of donor/acceptor-labeled GM1 on the surfaces of phospholipid bilayers are optimized to achieve high sensitivity.     

Superquenching as a detector for microsphere-based flow cytometric assays.

BACKGROUND: Fluorescent conjugated polymers display high fluorescence quantum yields and enhanced sensitivity to quenching (superquenching) by oppositely charged quenchers through energy or electron transfer. Fluorescent polymers and their quenchers are used in bead-based biosensor applications where the polymers are coated on particles. In this work, we investigate a detection method that utilizes superquenching on microspheres, which can be used for flow cytometric assays. METHODS: Microspheres were coated with the fluorescent cationic polyelectrolyte poly(p-phenylene-ethynylene) (PPE), and its superquenching by 9,10-anthraquinone-2,6-disulfonic acid (AQS) was examined by fluorometric methods in presence and in absence of a barrier to superquenching in the form of an anionic lipid bilayer. RESULTS: Flow cytometry detected superquenching of PPE on microspheres (MS-PPE) by AQS where high levels of reduction in fluorescence were observed. Adding different concentrations of AQS to MS-PPE yielded a Stern-Volmer quenching constant of 0.8x10(6) M-1. While forming an anionic lipid bilayer around the MS-PPE acted as a barrier to superquenching by AQS, disrupting the lipid bilayer allowed superquenching to take place. CONCLUSIONS: The sensitivity of flow cytometry in detecting fluorescence of microspheres and the amplified quenching sensitivity of fluorescent conjugated polymers both offer advantages over other fluorometric methods and conventional quenching detection. This study used superquenching of fluorescent polymers as a new tool in flow cytometry, thus combining the advantages offered by both method and detector. In addition, we employed the formation and the disruption of a supported lipid bilayer in mediating superquenching to offer new biosensing applications.     

Fringe-scan flow cytometry

We describe the development of a scanning flow cytometer capable of measuring the distribution of fluorescent dye along objects with a spatial resolution of 0.7 micron. The heart of this instrument, called a fringe-scan flow cytometer, is an interference field (i.e., a series of intense planes of illumination) produced by the intersection of two laser beams. Fluorescence profiles (i.e., records showing the intensity of fluorescence measured at 20 ns intervals) are recorded during the passage of objects through the fringe field. The shape of the fringe field is determined by recording light scatter profiles as 0.25 micron diameter microspheres traverse the field. The distribution of the fluorescent dye along each object passing through the fringe field is estimated from the recorded fluorescence profile using Fourier deconvolution. We show that the distribution of fluorescent dye along microsphere doublets and along propidium iodide stained human chromosomes can be determined accurately using fringe-scan flow cytometry. The accuracy of fringe-scan shape analysis was determined by comparing fluorescence profiles estimated from fringe-scan profiles for microspheres and chromosomes with fluorescence profiles for the same objects measured using slit-scan flow cytometry.     

Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging

Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50?nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20–30?nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication.     

Automated identification of subpopulations in flow cytometric list mode data using cluster analysis

The application of K-means (ISODATA) cluster analysis to flow cytometric data is described. The results of analyses of flow cytometric data for mixtures of fluorescent microspheres and samples of peripheral blood mononuclear cells are presented. A method for simultaneously displaying list mode data for any number of parameters, which had previously been applied to a continuous set of parameters such as multi-angle light scattering data, is used to present the results of cluster analysis on physically unrelated parameters; this method allows rapid evaluation of the success of subpopulation identification. The factors that influence automated identification of subpopulations are examined, and methods for determining optimal values for these factors are described.     

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres

BACKGROUND: Spectral interference (overlap) from phagocytosed green-yellow (GY) microspheres in the flow cytometric, red fluorescence emission measurement channel causes errors in quantifying damaged/dead alveolar macrophages by uptake of propidium iodide. METHODS: Particle burdens of uniform GY fluorescent microspheres phagocytosed by rat alveolar macrophages and the discrimination of damaged/dead cells as indexed by propidium iodide uptake were assessed with conventional and phase-sensitive flow cytometry. RESULTS: The fluorescence spectral emission from phagocytosed microspheres partly overlapped the propidium iodide red fluorescence emission and interfered with the measurement of damaged/dead cells when using conventional flow cytometry without subtractive compensation. This caused errors when estimating the percentage of nonviable, propidium iodide-positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in fluorescence lifetimes between the fluorescent microspheres and propidium iodide. Intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as that of the phagocytosed microspheres, also was eliminated in the phase-sensitive detection process. Because there was no detectable spectral interference of propidium iodide in the green fluorescence (phagocytosis) measurement channel, conventional fluorescence detection was employed. CONCLUSIONS: Phase-resolved, red fluorescence emission measurement eliminates spectral overlap errors caused by autofluorescent phagocytes that contain fluorescent microspheres in the analyses of propidium iodide uptake.Cytometry 39:45-55, 2000. Published 2000 Wiley-Liss, Inc.     

细菌群体感应信号分子的光电检测技术

群体感应（quorum sensing，QS）是一种依赖菌群密度的细菌交流系统。在探究细菌群体感应系统的调控机制中，对QS信号分子的鉴别和检测是不可或缺的环节，其对生命科学、药学等领域涉及细菌等微生物的相互作用、高效检测和作用机制解析等具有重要的参考意义。本文在总结不同类型细菌QS信号分子来源和结构的基础上，对QS信号分子的光电检测方法和技术进行了综述，重点对光电传感检测的敏感介质、传感界面、传感机制及测试效果进行探讨，同时关注了将微流控芯片分析技术应用于细菌QS信号分子原位监测的相关研究进展。     

High-temperature fluorescent in situ hybridization for detecting Escherichia coli in seawater samples, using rRNA-targeted oligonucleotide probes and flow cytometry

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46 degrees C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75 degrees C for 30 min) and (ii) two-step FISH (pretreatment in a 90 degrees C water bath for 5 min and a hybridizing step at 50 to 55 degrees C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).     

Imaging flow cytometry challenges the usefulness of classically used extracellular vesicle labeling dyes and qualifies the novel dye Exoria for the labeling of mesenchymal stromal cell–extracellular vesicle preparations

Background aimsExtracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed.MethodsBy combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia).ResultsThe authors’ analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types.ConclusionsOverall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors’ MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling.