使用密度梯度离心从人粪便中分离和纯化细菌胞外囊泡
Summary
本研究描述了一种 通过 密度梯度离心 （DGC） 分离和纯化从人类粪便中富集的细菌细胞外囊泡 （BEV） 的方法，从形态、粒径和浓度方面鉴定了 BEV 的物理特性，并讨论了 DGC 方法在临床和科学研究中的潜在应用。
Abstract
细菌细胞外囊泡 （BEV） 是来源于细菌的纳米囊泡，在细菌-细菌和细菌-宿主通讯中发挥积极作用，转移从亲本细菌遗传的蛋白质、脂质和核酸等生物活性分子。源自肠道微生物群的BEV在胃肠道内有影响，可以到达远处的器官，从而对生理学和病理学产生重大影响。探索源自人类粪便的BEV的类型、数量和作用的理论研究对于了解肠道微生物群中BEV的分泌和功能至关重要。这些研究也需要改进当前的BEV分离和纯化策略。
本研究通过建立自上而下和自下而上两种密度梯度离心（DGC）模式，优化了BEV的分离纯化过程。BEV的富集分布在分数6至8（F6-F8）中确定。根据颗粒形态、大小、浓度和蛋白质含量评估该方法的有效性。计算颗粒和蛋白质回收率，分析特异性标记物的存在，比较两种DGC模式的回收率和纯度。结果表明，自上而下离心模式具有较低的污染水平，并实现了与自下而上模式相似的回收率和纯度。7 h的离心时间足以达到108 / mg的粪便BEV浓度。
除粪便外，该方法可应用于其他体液类型，并根据成分和粘度的差异进行适当修改。综上所述，该详细可靠的方案将有助于BEV的标准化分离和纯化，从而为后续的多组学分析和功能实验奠定基础。
Introduction
肠道被广泛认为是人体内微生物群落最丰富的器官，超过 90% 的细菌参与定植和繁殖 1,2。大量证据表明，肠道微生物群调节肠道微环境，同时与远处器官的功能障碍相互作用，主要是通过受损的肠道屏障 3,4。越来越多的证据表明，肠道菌群的失衡与炎症性肠病 （IBD） 的进展之间存在相关性5,6，以及通过肠脑轴5,6,7,8 的认知障碍。细菌产生的细菌细胞外囊泡 （BEV） 在这些病理过程中起着重要作用。
BEV 是封装细菌衍生物的纳米级颗粒，直径范围为 20 至 400 nm。它们已被证明可以促进细菌与其宿主生物之间的相互作用 9,10。尽管它们不可见，但由于它们作为诊断生物标志物、治疗靶标和药物递送载体的广泛应用，这些颗粒越来越受到研究人员的关注11。人类粪便通常用作研究BEV的生物样本，主要来自肠道细菌，含有水、细菌、脂质、蛋白质、未消化的食物残渣和脱落的上皮细胞等的复杂混合物。复杂的粪便组成对BEV的分离和纯度提出了挑战，从而阻碍了对BEV的全面、客观和现实的分析。因此，尽量减少污染成分的干扰和提高纯电动汽车产量的有效策略已成为需要立即关注的关键问题。
现有的分离策略主要依赖于超高速离心 （UC）、密度梯度离心 （DGC） 和体积排阻色谱 （SEC）12、13、14、15、16、17 等技术。目前，DGC是BEV分离领域应用最广泛的方法之一，包括“自上而下”和“自下而上”两种沉降-浮动模式，由样品的初始加载位置决定。这些方法根据大小和密度差异将细胞外囊泡 （EV） 与其他组分区分开来，从而产生可变的纯度和回收率。先前的研究表明，单一方法策略不足以将 EV 与体液样本中的可溶性蛋白质充分分离，例如血液中的脂蛋白18 和尿液中的 Tamm-Horsfall 蛋白19。此外，真核细胞外囊泡 （EEV） 的大小分布通常与 BEV 的尺寸分布重叠，因此需要进一步改进方法学以优化 BEV 产量。因此，推进BEV的研究取决于开发有效的分离和纯化方法。值得注意的是，Tulkens等人[15]采用正交生物物理策略将粪便BEV与EEV分开，其中自下而上DGC模式的离心时间长达18 h。相比之下，本研究将其缩短至7 h，大大节省了梯度超速离心时间并简化了过程。
在本研究中，我们在优化的缓冲条件下，在以从低到极高的速度从低到极高的一系列差速离心速度富集BEV后，分离和纯化了采用两种DGC模式的粪便BEV。基于形态、粒径和浓度的评估表明，这种增强方法的性能值得称赞。这项研究可以作为未来研究的基础，将其应用扩展到更广泛的领域，并为人体内BEV的异质性提供见解。它还有助于BEV分离和分析技术的标准化。
Protocol
南方医科大学南方医院伦理委员会批准了这项研究，该研究是在参与者知情同意的情况下进行的。本文采用的所有方法均符合国际人类微生物组标准（IHMS：http://www.microbiome-standards.org/）提供的标准操作指南。所有随后的液体处理程序都必须在生物安全柜或超洁净工作台内进行。 1. 粪便样本的采集和分装 分发粪便采样器、密封袋和冰盒，并向参与者提供有…
Representative Results
确定富含BEV的馏分的分布为了确定富含细菌胞外囊泡（BEV）的组分的分布，建立了一个空白对照以测量OD 340nm处的吸光度值，并根据测量值和碘克沙醇指南计算每个组分的密度（步骤8.1）。 表 2 显示了密度结果，表明 F4 至 F9 级分的密度在通常与细胞外囊泡相关的范围内。这一发现表明，大多数BEV在这些部分中被分离出来，导致将F4-F9定义为BEV的粗略范围。采用透射电?…
Discussion
细菌细胞外囊泡 （BEV） 是细菌分泌的脂质双层纳米颗粒，携带丰富的蛋白质、脂质、核酸和其他生物活性分子，有助于介导细菌的功能效应20。源自肠道的BEV已被证实与炎症性肠病、克罗恩病和结直肠癌等疾病的发展有关，还会影响一般代谢并介导认知功能受损4,16,17,20,21,22,23,24,25,26 </su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究得到了 国家杰出青年科学基金（82025024）、国家自然科学基金重点项目（82230080）; 国家重点研发计划（2021YFA1300604）;国家自然科学基金（81871735、82272438、82002245）;广东省杰出青年自然科学基金（2023B1515020058）; 广东省自然科学基金（2021A1515011639）; 山东省自然科学基金国家重大基础研究发展计划（ZR2020ZD11）; 博士后科学基金（2022M720059）;南方医科大学南方医院优秀青年发展计划（2022J001）。
Materials
| 1 % (w/v) glutaraldehyde (prepared from 2.5 % stock solution in deionized water) | ACMEC | AP1126 | Morphological observation for BEVs using TEM at Step 8.3.3 |
| 1 % (w/v) methylcellulose (prepared from original powder in deionized water) | Sigma-Aldrich | M7027 | Morphological observation for BEVs using TEM at Step 8.3.6 |
| 1.5 % (w/v) uranyl acetate (prepared from original powder in deionized water) | Polysciences | 21447-25 | Morphological observation for BEVs using TEM at Step 8.3.5 |
| 1000 μL, 200 μL, 10 μL Pipette | KIRGEN | KG1313, KG1212, KG1011 | Transfer the solution |
| 5 % (w/v) bovine serum albumin solution (prepared from the original powder in TBST buffer) | Fdbio science | FD0030 | Used in western blotting for blocking at Step 8.5.6 |
| 5 × loading buffer | Fdbio science | FD006 | Used in western blotting and Coomassie brilliant blue stain at Step 8.5.1 |
| 75 % (v/v) alcohol | LIRCON | LIRCON-500 mL | Surface disinfection |
| 96-well plate | Rar | A8096 | Measure the absorbance values |
| Anti-Calnexin antibody | Abcam | ab92573 | Western blotting (Primary Antibody) |
| Anti-CD63 antibody | Abcam | ab134045 | Western blotting (Primary Antibody) |
| Anti-CD9 antibody | Abcam | ab236630 | Western blotting (Primary Antibody) |
| Anti-Flagellin antibody | Sino Biological | 40067-MM06 | Western blotting (Primary Antibody) |
| Anti-Integrin beta 1 antibody | Abcam | ab30394 | Western blotting (Primary Antibody) |
| Anti-LPS antibody | Thermo Fisher | MA1-83152 | Western blotting (Primary Antibody) |
| Anti-LTA antibody | Thermo Fisher | MA1-7402 | Western blotting (Primary Antibody) |
| Anti-OmpA antibody | CUSABIO | CSB-PA359226ZA01EOD, https://www.cusabio.com/ | Western blotting (Primary Antibody) |
| Anti-Syntenin antibody | Abcam | ab133267 | Western blotting (Primary Antibody) |
| Anti-TSG101 antibody | Abcam | ab125011 | Western blotting (Primary Antibody) |
| Autoclave | ZEALWAY | GR110DP | Sterilization for supplies and mediums used in the experiment |
| Balance | Mettler Toledo | AL104 | Balance the tube sample-loaded with PBS |
| Bicinchoninic acid assay | Fdbio science | FD2001 | Measure protein content of BEVs at Step 8.2 |
| BioRender | BioRender | https://app.biorender.com | Make the schematic workflow of BEVs isolation and purification showed in Figure 1 |
| Biosafety cabinet | Haier | HR1200- II B2 | Peform the procedures about feces sample handling |
| Centrifuge 5810 R; Rotor F-34-6-38 | Eppendorf | 5805000092; 5804727002, adapter: 5804774000 | Preprocess for BEVs (Step 3) |
| Chemiluminescence Apparatus | BIO-OI | OI600SE-MF | Used in western blotting for signal detection at Step 8.5.12 |
| Cytation 5 | BioTek | F01 | Microplate detector for measuring the absorbance (Step 8.1) and fluorescence (Figure 6) values |
| Dil-labled low density lipoprotein | ACMEC | AC12038 | Definition of distribution of interfering components |
| Electrophoresis equipment | Bio-rad | 1658033 | Used in western blotting for protein separation and transfer at Step 8.5.2, 8.5.3, 8.5.5 |
| Enhanced Chemiluminescence kit HRP | Fdbio science | FD8020 | Used in western blotting for signal detection at Step 8.5.12 |
| Escherichia coli | American Type Culture Collection | ATCC8739 | Isolate BEVs as a positive control. Protocol: Dissolve 25 g of the LB powder in 1 L deionized water, and autoclave. Transfer the 800 μL of preserved Escherichia coli into the medium. Cultivate at 37 °C in the incubator shaker. Then centrifuge at 3, 000 × g for 20 min at 4 °C, 12, 000 × g for 30 min at 4 °C, filter the supernatant through 0.22 μm membrane, and perform ultra-speed centrifugation at 160, 000 × g for 70 min at 4 °C. Pellet defined as crude BEVs from Escherichia coli was suspended in 1.2 mL PBS (Step 3, 4). |
| Falcon tubes 50 mL | KIRGEN | KG2811 | Preprocess for BEVs (Step 3) |
| Feto Protein Staining Buffer | Absci | ab.001.50 | Coomassie brilliant blue staining at Step 8.5.4 |
| Filter paper | Biosharp | BS-TFP-070B | Morphological observation for BEVs using TEM at Step 8.3 (Blotting the solution) |
| Formvar/Carbon supported copper grids | Sigma-Aldrich | TEM-FCF200CU50 | Morphological observation for BEVs using TEM at Step 8.3 |
| HEPES powder | Meilunbio | MB6078 | Prepare iodixanol buffers with different concentrations for density gradient centrifugation |
| HRP AffiniPure Goat Anti-Mouse IgG (H+L) | Fdbio science | FDM007 | Western blotting (Secondary Antibody) |
| HRP AffiniPure Goat Anti-Rabbit IgG (H+L) | Fdbio science | FDR007 | Western blotting (Secondary Antibody) |
| Incubator shaker | Qiangwen | DHZ-L | Cultivate Escherichia coli |
| Kimwipes™ Delicate Task Wipes | Kimtech Science | 34155 | Wipe the inner wall of the ultracentrifuge tube at Step 4.15 |
| LB broth | Hopebio | HB0128 | Cultivate Escherichia coli |
| Low temperature freezer (-80 °C) | Haier | DW-86L338J | Store the samples |
| Methanol | Alalddin | M116118 | Used in western blotting for activating PVDF membrane at Step 8.5.5 |
| Micro tubes 1.5 mL | KIRGEN | KG2211 | Recover fractions after density gradient centrifugation |
| Micro tubes 2 mL | KIRGEN | KG2911 | Recover fractions after density gradient centrifugation |
| Micro tubes 5 mL | BBI | F610888-0001 | Recover fractions after density gradient centrifugation |
| Microplate reader | Thermo Fisher | Multiskan MK3 | Measure protein content of BEVs at Step 8.2 |
| Millipore filter 0.22 μm | Merck millipore | SLGP033RB | Filtration sterilization; Material: polyethersulfone, PES |
| NaCl | GHTECH | 1.01307.040 | Density gradient centrifugation solution |
| NaOH | GHTECH | 1.01394.068 | Density gradient centrifugation solution (pH adjustment) |
| Optima™ XPN-100 | Beckman Coulter | A94469 | Ultracentrifugation for BEVs isolation at Step 4, 7 |
| OptiPrep™ | Serumwerk Bernburg AG | 1893 | Density gradient centrifugation stock solution |
| Orbital Shaker | Youning | CS-100 | Dissolve feces at Step 2 |
| Phosphate buffered saline | Procell | PB180327 | Dissolve feces at Step 2 |
| Pipettor | Eppendorf | 3120000267, 3120000259 | Transfer the solution |
| Plastic pasteur pipette | ABCbio | ABC217003-4 | Remove supernatant in preprocessing at Step 3.4 |
| Polyvinylidene difluoride (PVDF) membranes | Millipore | ISEQ00010, IPVH00010 | Used in western blotting for protein transfer at Step 8.5.5 |
| Prefabricated polyacrylamide gel, 4–20% 15 Wells | ACE | F15420Gel | Used in western blotting for protein separation at Step 8.5.2, 8.5.3 |
| Primary antibody diluent | Fdbio science | FD0040 | Used in western blotting at Step 8.5.8 |
| Protein ladder | Fdbio science | FD0672 | Used in western blotting and Coomassie brilliant blue stain at Step 8.5 |
| Rapid protein blotting solution | UBIO | UW0500 | Used in western blotting for protein transfer at Step 8.5.5 |
| Rotor SW 32 Ti Swinging-Bucket Rotor | Beckman Coulter | 369650 | Ultracentrifugation for BEVs isolation at Step 4, 7 |
| Syringe 20 mL, 50 mL | Jetway | ZSQ-20ML, YCXWJZSQ-50 mL | Transfer buffers amd remove supernatant in preprocessing |
| TBS powder | Fdbio science | FD1021 | Used in western blotting at Step 8.5 |
| Transmission electron microscope (TEM) | Hitachi | H-7650 | Morphological observation for BEVs at Step 8.3 |
| Tween-20 | Fdbio science | FD0020 | Used in western blotting at Step 8.5 |
| Ultracentrifuge tube | Beckman | 326823, 355642 | Ultracentrifugation for BEVs isolation at Step 4, 7 |
| Ultra-clean bench | AIRTECH | SW-CJ-2FD | Peform the procedures about liquid handling |
| Water bath | Bluepard | CU600 | Used for measuring protein content of BEVs at Step 8.2.5 |
| ZetaView | Particle Metrix | S/N 21-734, Software ZetaView (version 8.05.14 SP7) | Nanoparticle tracking analysis (NTA) for measuring the particle size and concentrarion of BEVs at Step 8.4 |
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Cite This Article
Xue, Y., Huang, X., Ou, Z., Wu, Y., Li, Q., Huang, X., Wen, M., Yang, Y., Situ, B., Zheng, L. Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation. J. Vis. Exp. (199), e65574, doi:10.3791/65574 (2023).