免疫细胞衍生细胞外囊泡的表征及研究功能对细胞环境的影响
Summary
本报告强调了从微胶质或血液巨噬细胞分离细胞外囊泡（EV）的时间顺序要求。微胶质衍生的EV被评价为神经外生长的调节器，而血液巨噬细胞衍生的EV在体外检测中控制C6胶质瘤细胞入侵时被研究。目标是更好地了解这些EV功能作为免疫中介器在特定微环境中。
Abstract
中枢神经系统（CNS）的神经炎状态在生理和病理状况中起着关键作用。微胶质，大脑中的常驻免疫细胞，有时渗入骨髓衍生的巨噬细胞（BMDM），调节其微环境在中枢神经系统的炎症特征。现在人们承认，免疫细胞的细胞外囊泡（EV）种群充当免疫中介。因此，它们的收集和隔离对于识别其内容，但也评估其对受体细胞的生物影响也很重要。本数据强调了从微胶质细胞或血液巨噬细胞（包括超中心化和大小排除色谱（SEC）步骤中分离EV的时间顺序要求。非靶向蛋白组分析允许将蛋白质特征验证为EV标记，并特征为生物活性EV含量。微胶质衍生的EV也功能地用于神经元的主要培养，以评估它们作为中微子生长中免疫中介的重要性。结果表明，微胶质衍生的EV有助于促进体外微子的生长。同时，血液巨噬细胞衍生的EV在C6胶质瘤细胞的球体培养物中作为免疫中介器，结果表明这些EV在体外控制胶质瘤细胞入侵。本报告强调了评估EV介导免疫细胞功能的可能性，但也了解这种通信的分子基础。这种破译可以促进使用天然囊泡和/或体外制备治疗性囊泡，以模仿中枢神经系统病理学微环境中的免疫特性。
Introduction
许多神经病理学与神经炎症状态有关，这是一种日益被考虑的复杂机制，但由于免疫过程多种多样，并且依赖于细胞环境，因此仍难以理解。事实上，中枢神经系统疾病没有系统地涉及相同的激活信号和免疫细胞群，因此亲炎反应很难作为病理的原因或后果来评价。大脑常驻巨噬细胞称为”微胶质”，似乎在神经和免疫系统之间的接口1。微胶质有一个骨髓起源，从原始造物病期间的蛋黄囊派生，以殖民大脑，而外周巨噬细胞从胎儿肝脏在决定性的造物形成过程中，成为外周巨噬细胞2。微胶质细胞与神经元和神经元衍生的胶质细胞（如星形细胞和寡核糖3）通信。最近的几项研究表明，微胶质在中枢神经系统发育和成人组织平衡过程中，以及与神经退行性疾病44、55相关的炎症状态中，都涉及神经元可塑性。否则，血脑屏障的完整性可能会在其他中枢神经系统疾病中受损。免疫反应，特别是在胶质细胞瘤多肠癌中，不仅由微胶质细胞支持，因为血脑屏障通过血管生成过程和淋巴血管的存在6，77重组。因此，在整个肿瘤依赖性血管生成机制8中，脑肿瘤中出现了一个大的骨髓衍生巨噬细胞（BMDM）。癌细胞对渗透的BMDM有显著影响，导致免疫抑制特性和肿瘤生长9。因此，由于细胞起源和激活信号多种多样，免疫细胞与其大脑微环境之间的通信是难以理解的。因此，在生理条件下，对免疫细胞相关分子特征的功能进行鉴定是很有趣的。在这方面，通过释放细胞外囊泡（EV），可以研究免疫细胞和细胞微环境之间的细胞-细胞通信。
在调节健康及病理状况的,免疫功能方面，EV正研究得越来越多。可以考虑两个种群，外体和微囊。它们呈现不同的生物发生和大小范围。外泌体是直径±30~150nm的囊泡，由内体系统生成，在多孔体（MVB）与等离子膜融合期间分泌。微囊直径约为100~1000纳米，由细胞等离子膜14外萌芽产生。由于外泌体与微囊歧视仍难以根据大小和分子模式来实现，因此本文仅使用”EV”一词。CNS的EV相关通信代表了一种祖先机制，因为研究表明它们与无脊椎动物物种（包括线虫、昆虫或阴叶15、16）,16有牵连。此外，结果显示，EV可以与不同物种的细胞通信，表明这种机制是一个关键锁系统，首先基于囊泡和受体细胞之间的表面分子识别，然后允许接受调子16，17。16,事实上，EV含有许多分子，如蛋白质（如酶、信号转导、生物成因）、脂质（如神经酰胺、胆固醇）或核酸（例如DNA、mRNA或miRNA），作为受体细胞活动的直接或间接调节剂14。这就是为什么方法学研究也进行免疫细胞分离EV和充分表征他们的蛋白质特征18，19。18,
最早的研究表明，在Wnt3a-或血清素依赖活化20，21,21之后，从原培养大鼠微胶质中释放外体是一种诱导机制。在CNS的功能中，微胶质衍生的EV调节突触神经质释放通过神经元的预显终端促进神经元兴奋性控制22，23。22,微胶衍生的EV也可以传播细胞因子介导的炎症反应在大大脑区域24，25。24,重要的是，收费型受体家族的多样化配体可能会激活微胶质26中特定EV的生产。例如，体外研究表明，LPS激活的微胶质BV2细胞系产生微分EV含量，包括亲炎细胞因子27。因此，通过自身的EV种群，包括EV对受体细胞的影响和EV内容的识别，可以评估中枢神经系统中免疫细胞亚群的功能多样性、微胶质和渗透性BMDM。
我们之前描述了在微胶质和BMDM衍生的EV分离16，19,19后的功能特性的方法。在本报告中，我们建议独立评估微胶质衍生的EV对神经质外生长的影响，以及巨噬细胞衍生的EV对胶质瘤细胞聚合控制的影响。本研究还提出了对EV分数进行广泛的蛋白质组分析，以验证EV分离程序，并识别生物活性蛋白特征。EV内容的有益效果和分子破译可以帮助它们可能操纵，并作为脑疾病的治疗剂使用。
Protocol
1. 微胶质/巨噬细胞的主要文化 微胶质的主要文化 培养商业大鼠原生微胶质（2 x 106细胞）（见材料表）在Dulbecco的改良鹰介质（DMEM）中补充10%无异体血清、100 U/mL青霉素、100微克/mL链球菌素和9.0克/L葡萄糖，37°C和5%CO2 。2 收集48小时培养后的条件介质，然后进行 EV 隔离。 巨噬细胞的主要培养 ?…
Representative Results
将生物效应归因于细胞外囊泡 （EV） 的主要挑战之一是能够将 EV 与整个培养介质隔离开来。在本报告中，我们提出了一种使用超中心（UC）和大小排除色谱（SEC）的方法，该方法与蛋白质特征的大规模分析相结合，以验证EV标记物并识别生物活性化合物。巨噬细胞或微胶质衍生的EV分别在24小时或48小时培养后与条件介质分离（图1）。 <p class="jove_content" fo:keep-together.within-p…
Discussion
中枢神经系统（CNS）是一个复杂的组织，细胞到细胞的通信调节平衡所需的正常神经元功能30。EV现已被广泛研究，并被描述为细胞到细胞通信的重要分子货物31。他们特别向受体细胞提供一种调子鸡尾酒，从而影响其在健康和病理条件下的功能32。最近的研究表明，EV在中枢神经系统24、33、3433,34…
Disclosures
The authors have nothing to disclose.
Acknowledgements
提交的作品得到了全国教育促进中心、全国教育与教育协会的支持。我们感谢BICeL-校园科学城设施获得仪器和技术建议。我们感谢让-帕斯卡尔·吉梅诺、苏莱曼·阿布卢亚德和伊莎贝尔·富尼耶为大众测量学援助。我们感谢塔尼娜·阿拉伯人、克里斯特尔·范坎普、弗朗索瓦丝·勒马雷克-克罗克、雅科波·维齐奥利和皮埃尔-埃里克·萨乌蒂埃对科学和技术发展所作的重大贡献。
Materials
| 12% Mini-PROTEAN TGX Precast Protein Gels | Bio-rad | 4561045EDU | |
| Acetonitrile | Fisher Chemicals | A955-1 | |
| Amicon 50 kDa centrifugal filter | Merck | UFC505024 | |
| Ammonium bicarbonate | Sigma-Aldrich | 9830 | |
| HSP90 α/β antibody (RRID: AB_675659) | Santa-cruz | sc-13119 | |
| B27 Plus supplement | Gibco | A3582801 | |
| BenchMixer V2 Vortex Mixer | Benchmark Scientific | BV1003 | |
| Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford) | Bio-Rad | 5000006 | |
| C18 ZipTips | Merck Millipore | ZTC18S096 | |
| C6 rat glioma cell | ATCC | ATCC CCL-107 | |
| Canonical tubes | Sarstedt | 62.554.002 | |
| Centrifuge | Eppendorf | 5804000010 | |
| CO2 Incubator | ThermoFisher | ||
| Confocal microscope LSM880 | Carl Zeiss | LSM880 | |
| Cover glass | Marienfeld | 111580 | |
| Culture Dish (60 mm) | Sarstedt | 82.1473 | |
| Dithiothreitol | Sigma-Aldrich | 43819 | |
| DMEM | Gibco | 41966029 | |
| EASY-nLC 1000 Liquid Chromatograph | ThermoFisher | ||
| Electron microscope JEM-2100 | JEOL | ||
| Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid | Sigma-Aldrich | 03777-10G | |
| Ethylenediaminetetraacetic acid | Sigma-Aldrich | ED-100G | |
| Exo-FBS | Ozyme | EXO-FBS-50A-1 | Exosome depleted FBS |
| ExoCarta database (top 100 proteins of Evs) | http://www.exocarta.org/ | ||
| Fetal Bovine Serum | Gibco | 16140071 | |
| Fetal Horse Serum | Biowest | S0960-500 | |
| Filtropur S 0.2 | Sarstedt | 83.1826.001 | |
| Fisherbrand Q500 Sonicator with Probe | Fisherbrand | 12893543 | |
| FlexAnalysis | Brucker | ||
| Fluorescence mounting medium | Agilent | S3023 | |
| Formic Acid | Sigma-Aldrich | 695076 | |
| Formvar-carbon coated copper grids | Agar scientific Ltd | AGS162-3 | |
| Glucose | Sigma-Aldrich | G8769 | |
| Glutaraldehyde | Sigma-Aldrich | 340855 | |
| Hoechst 33342 | Euromedex | 17535-AAT | |
| Idoacetamide | Sigma-Aldrich | I1149 | |
| InstantBlue Coomassie Protein Stain | Expedeon | ISB1L | |
| Invert light microscope CKX53 | Olympus | ||
| L-glutamine | Gibco | 25030-024 | |
| LabTek II 8 wells | Nunc | 154534 | |
| Laemmli 2X | Bio-Rad | 1610737 | |
| Laminin | Corning | 354232 | |
| MaxQuant software (proteins identification software) | https://maxquant.net/maxquant/ | ||
| MBT Polish stell | Brucker | 8268711 | |
| MEM 10X | Gibco | 21090-022 | |
| Methylcellulose | Sigma-Aldrich | M6385-100G | |
| MiliQ water | Merck Millipore | ||
| Milk | Regilait | REGILAIT300 | |
| Mini PROTEAN Vertical Electrophoresis Cell | Bio-Rad | 1658000FC | |
| MonoP FPLC column | GE Healthcare | no longer available | |
| Nanosight NS300 | Malvern Panalytical | NS300 | |
| NanoSight NTA software v3.2 | Malvern Panalytical | ||
| NanoSight syringe pump | Malvern Panalytical | ||
| Neurobasal | Gibco | 21103-049 | |
| Nitrocellulose membrane | GE Healthcare | 10600007 | |
| Nonidet P-40 | Fluka | 56741 | |
| Nunc multidish 24 wells | ThermoFisher | 82.1473 | |
| Paraformaldehyde | Electro microscopy Science | 15713 | |
| PC-12 cell line | ATCC | ATCC CRL-1721 | |
| Penicillin-Streptomycin | Gibco | 15140-122 | |
| Peptide calibration mix | LaserBio Labs | C101 | |
| Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 115-035-003 | |
| Perseus software (Processing of identified proteins) | https://maxquant.net/perseus/ | ||
| Phalloidin-tetramethylrhodamine conjugate | Santa-cruz | sc-362065 | |
| Phenylmethanesulfonyl fluoride | Sigma-Aldrich | 78830 | |
| Phosphate Buffer Saline | Invitrogen | 14190094 | no calcium, no magnesium |
| pluriStrainer M/ 60 µm | pluriSelect | 43-50060 | |
| Poly-D-lysine | Sigma-Aldrich | P6407 | |
| Polycarbonate centrifuge tubes | Beckman Coulter | 355651 | |
| Protease Inhibitor | Sigma-Aldrich | S8830-20TAB | |
| PureCol | Cell Systems | 5005 | |
| Q-Exactive mass spectrometer | ThermoFisher | ||
| rapifleX mass spectrometer | Brucker | ||
| Rat cortical neurons | Cell Applications | R882N-20 | Cell origin : Derived from cerebral cortices of day 18 embryonic Sprague Dawley rat brains |
| Rat Macrophage & Microglia Culture Medium | Cell Applications | R620K-100 | Cell orgin : Normal healthy Rat bone marrow |
| Rat primary macrophages | Cell Applications | R8818-10a | |
| Rat primary microglia | Lonza | RG535 | |
| Sepharose CL-2B | GE Healthcare | 17014001 | |
| Sequencing Grade Modified Trypsin | Promega | V5111 | |
| Slide | Dustsher | 100204 | |
| Sodium Chloride | Scharlau | SO0227 | |
| Sodium Dodecyl Sulfate | Sigma-Aldrich | L3771 | |
| Sodium Fluoride | Sigma-Aldrich | S7920-100G | |
| Sodium hydroxide | Scharlab | SO0420005P | |
| Sodium pyrophosphate | Sigma-Aldrich | S6422-100G | |
| SpeedVac Vacuum Concentrator | ThermoFisher | ||
| String software (functional protein association networks) | https://string-db.org/ | ||
| SuperSignal West Dura extended Duration Substrate | ThermoFisher | 34075 | |
| Syringe 1.0 mL | Terumo | 8SS01H1 | |
| Trans-Blot SD Semi-Dry Transfer cell | Bio-Rad | 1703940 | |
| Trifluoroacetic acid | Sigma-Aldrich | T6508 | |
| Tris | Interchim | UP031657 | |
| Tris-Glycine | Euromedex | EU0550 | |
| Tween 20 | Sigma-Aldrich | P2287 | |
| Ultracentrifuge | Beckman Coulter | A95765 | |
| Ultracentrifuge Rotor 70.1 Ti | Beckman Coulter | 342184 | |
| Uranyl acetate | Agar Scientific Ltd | AGR1260A | |
| Whatman filter paper | Sigma-Aldrich | WHA10347510 | |
| α-Cyano-4-hydroxycinnamic acid | Sigma-Aldrich | C2020-25G |
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Cite This Article
Lemaire, Q., Duhamel, M., Raffo-Romero, A., Salzet, M., Lefebvre, C. Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment. J. Vis. Exp. (160), e60118, doi:10.3791/60118 (2020).