从血浆和实体组织中分离和分析可追溯和功能化的细胞外囊泡
1State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology,The Fourth Military Medical University, 2Department of Orthodontics, School of Stomatology,The Fourth Military Medical University, 3Department of VIP Dental Care, School of Stomatology,The Fourth Military Medical University, 4School of Basic Medicine,The Fourth Military Medical University, 5College of Life Science,Northwest University, 6Xi’an Institute of Tissue Engineering and Regenerative Medicine
Summary
本协议描述了一种从外周血和实体组织中提取细胞外囊泡的方法，随后对表面抗原和蛋白质货物进行分析。
Abstract
循环和组织驻留的细胞外囊泡（EVs）作为新型治疗诊断生物标志物代表了有希望的靶标，它们成为维持机体稳态和广泛疾病进展的重要参与者。虽然目前的研究重点是具有内体起源的内源性外泌体的特征，但从质膜中起泡的微囊泡在健康和疾病中越来越受到关注，其特征是丰富的表面分子概括了亲本细胞的膜特征。在这里，提出了一种基于差速离心的可重复程序，用于从血浆和实体组织（例如骨骼）中提取和表征EV。该协议进一步描述了电动汽车表面抗原和蛋白质货物的后续分析，因此可以追溯其衍生物，并识别与潜在功能相关的组件。该方法将有助于在生物学、生理学和病理学研究中对 EV 进行相关、功能和机理分析。
Introduction
已经提出细胞外囊泡（EV）来定义细胞释放的脂质双层封闭的细胞外结构1，其在各种生理和病理事件中起重要作用2。健康细胞释放的EV大致可分为两大类，即通过细胞内内吞运输途径3形成的外泌体（或小EV）和细胞质膜向外出芽形成的微囊泡（或大EV）4。虽然许多研究集中在体外5中从培养细胞中收集的EV的功能，但来自循环或组织的EV更加复杂和异质，其优点是反映了体内生物体的真实状态6。此外，几乎所有种类的组织都可以在体内产生EV，这些EV可以作为组织内的信使或通过各种体液（尤其是外周血）转移，以促进全身交流7。循环和组织中的电动汽车也是疾病诊断和治疗的目标8。
近年来，外泌体的研究深入，微囊泡也具有重要的生物学功能，无需超速离心即可轻松提取，从而促进了基础和临床研究9。值得注意的是，关于从循环和组织中分离的EV的一个关键问题是它们来自不同的细胞类型10。由于微囊泡从质膜中吹出并具有丰富的细胞表面分子9，因此使用亲本细胞膜标记物来鉴定这些EV的细胞起源是可行的。具体而言，流式细胞术（FC）技术可用于检测膜标志物。此外，研究人员可以分离电动汽车并根据功能货物进行进一步分析。
本协议为从体内样品中提取和表征EV提供了全面的程序。EV通过差速离心分离，EV的表征包括通过纳米颗粒跟踪分析（NTA）和透射电子显微镜（TEM）进行形态学鉴定，通过FC进行原产地分析，以及通过蛋白质印迹进行蛋白质货物分析。以小鼠的血浆和上颌骨为代表。研究人员可以参考其他来源的电动汽车的该协议并进行相应的修改。
Protocol
动物实验按照《第四军医大学机构动物护理使用委员会指南》和《ARRIVE指南》进行。在本研究中，使用8周龄的C57Bl / 6小鼠（对雌性或雄性都没有偏好）。分离血浆和组织EV所涉及的步骤如图 1所示。血浆以代表描述EV从体液中分离的过程。以上颌骨为代表，解释EV与实体组织的分离过程。 1. 血浆和上颌骨样本的制备 按照以下步骤制备?…
Representative Results
根据实验工作流程，可以从外周血和实体组织中提取EV（图1）。8周龄小鼠的上颌骨约为0.1±0.05g，可以从小鼠中收集约300μL血浆。按照协议步骤，可以分别收集0.3 mg和3 μg的EV。通过TEM和NTA分析，EV的典型形态特征是直径范围为50-300nm的圆形杯形膜囊泡（图2）。FC可以在有用的对照下检测膜染料标记的EV，FC分析显示了EV上表达的特定膜标志物的百分比，这…
Discussion
在研究电动汽车的特性、命运和功能时，隔离高产量和低污染的电动汽车至关重要。有多种方法可以提取 EV，例如密度梯度离心 （DGC）、体积排阻色谱 （SEC） 和免疫捕获测定4，20。这里使用了最常用的方法之一，差速离心;这样做的优点是不耗时，它可以产生高产量的EV，易于从有限的样品中分离出来，并且能够根据所使用的样品源修改方法。为了分?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家自然科学基金（32000974，81870796，82170988和81930025）和中国博士后科学基金（2019M663986和BX20190380）的资助。我们感谢国家基础医学实验教学示范中心（AMFU）的帮助。
Materials
| 4% paraformaldehyde | Biosharp | 143174 | Transmission electron microscope |
| Alexa fluor 488 anti-goat secondary antibody | Yeason | 34306ES60 | Flow cytometry |
| Alexa fluor 488 anti-rabbit secondary antibody | Invitrogen | A11008 | Flow cytometry |
| Anti-CD18 antibody | Abcam | ab131044 | Flow cytometry |
| Anti-CD81 antibody | Abcam | ab109201 | Western blot |
| anti-CD9 antibody | Huabio | ET1601-9 | Western blot |
| Anti-Mitofilin antibody | Abcam | ab110329 | Western blot |
| APOA1 Rabbit pAb | Abclone | A14211 | Western blot |
| BCA protein assay kit | TIANGEN | PA115 | Western blot |
| BLUeye Prestained Protein Ladder | Sigma-Aldrich | 94964-500UL | Western blot |
| Bovine serum albumin | MP Biomedical | 218072801 | Western blot |
| Caveolin-1 antibody | Santa Cruz Biotechnology | sc-53564 | Western blot |
| CellMask Orange plasma membrane stain | Invitrogen | C10045 | Flow cytometry |
| Chemiluminescence | Amersham Biosciences | N/A | Western blot |
| Curved operating scissor | JZ Surgical Instrument | J21040 | EV isolation |
| Electronic balance | Zhi Ke | ZK-DST | EV isolation |
| Epoch spectrophotometer | BioTek | N/A | Western blot |
| Eppendorf tubes | Eppendorf | 3810X | EV isolation |
| Flotillin-1 antibody | PTM BIO | PTM-5369 | Western blot |
| Gel imaging system | Tanon | 4600 | Western blot |
| Golgin84 | Novus | nbp1-83352 | Western blot |
| Grids – Formvar/Carbon Coated – Copper 200 mesh | Polysciences | 24915 | Transmission electron microscope |
| Heparin Solution | StemCell | 7980 | EV isolation |
| Liberase Research Grade | Sigma-Aldrich | 5401127001 | EV isolation |
| Microscopic tweezer | JZ Surgical Instrument | JD1020 | EV isolation |
| NovoCyte flow cytometer | ACEA | N/A | Flow cytometry |
| Omni-PAGE Hepes-Tris Gels Hepes 4~20%, 10 wells | Epizyme | LK206 | Western blot |
| OSCAR(D-19) antibody | Santa Cruz Biotechnology | SC-34235 | Flow cytometry |
| PBS (2x) | ZHHC | PW013 | Western blot |
| Pentobarbital sodium | Sigma-Aldrich | 57-33-0 | Anesthetization |
| Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) | Jacson | 115-035-003 | Western blot |
| Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) | Jacson | 111-035-003 | Western blot |
| Phosphotungstic acid | RHAWN | 12501-23-4 | Transmission electron microscope |
| PKM2(d78a4) xp rabbit mab | Cell Signaling | 4053t | Western blot |
| Polyethylene (PE) film | Xiang yi | 200150055 | Transmission electron microscope |
| Polyvinylidene fluoride membranes | Roche | 3010040001 | Western blot |
| Protease inhibitors | Roche | 4693132001 | Western blot |
| Recombinant anti-PGD antibody | Abcam | ab129199 | Western blot |
| RIPA lysis buffer | Beyotime | P0013 | Western blot |
| SDS-PAGE loading buffer (5x) | Cwbio | CW0027S | Western blot |
| Size beads | Invitrogen | F13839 | Flow cytometry |
| Tabletop High-Speed Micro Centrifuges | Hitachi | CT15E | EV isolation |
| Transmission electron microscope | HITACHI | H-7650 | Transmission electron microscope |
| Tween-20 | MP Biomedicals | 19472 | Western blot |
| Vortex Mixer Genie | Scientific Industries | SI0425 | EV isolation |
| ZetaView BASIC NTA – Nanoparticle Tracking Video Microscope PMX-120 | Particle Metrix | N/A | Nanoparticle tracking analysis |
| α-Actinin-4 Rabbit mAb | Abclone | A3379 | Western blot |
| β-actin | Cwbio | CW0096M | Western blot |
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Cao, Y., Qiu, J., Chen, D., Li, C., Xing, S., Zheng, C., Liu, X., Jin, Y., Sui, B. Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues. J. Vis. Exp. (188), e63990, doi:10.3791/63990 (2022).