使用密度梯度离心从人粪便中分离和纯化细菌胞外囊泡

摘要

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。

摘要

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。

引言

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....

研究方案

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

1. 分发粪便采样器、密封袋和冰盒，并向参与者提供有关如何获取和保存样本的全面说明。
2. 指示每个参与者使用提供的采样器收集他们的粪便样本，并在 24 小时窗口内将其以 4 °C 的温度运送到实验室。
3. 在实验室收到后，使用无菌勺将少于 3.5 g 的粪便分装到预先称重的 50 mL 离心管中。在试管上记下粪便样本的重量，以便进行后续的预处理程序。
   暂停点：如果无法立即处理样品，请在适当记号后将样品分配在-80°C下长期储存。

2. 粪便样品制备

1. 在4°C下冷却500mL磷酸盐缓冲盐水（PBS）。 使用 50 mL 注射器通过 0.22 μm 聚醚砜 （PES） 过滤器将预冷的 PBS 过滤到 10 个 50 mL 离心管中。
2. 将两个 50 mL 试管放在冰上，每个试管含有 3.5 g 粪便样品。向每个试管中加入 35 mL P....

讨论

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

材料

Name	Company	Catalog Number	Comments
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