肝癌组织来源于小细胞外囊泡的富集方法
Summary
在这里，我们描述了通过优化的差分超速离心方法富集来自肝癌组织的小细胞外囊泡。
Abstract
来源于组织的小细胞外囊泡（sEV）可以反映源细胞的功能状态和组织间质空间的特征。这些sEVs的高效富集是研究其生物学功能的重要前提，也是临床检测技术和治疗载体技术发展的关键。很难从组织中分离出sEV，因为它们通常受到严重污染。本研究提供了一种从肝癌组织中快速富集高质量sEV的方法。该方法涉及一个四步过程：消化酶（胶原酶D和DNase Ι）与组织的孵育，通过70μm细胞过滤器过滤，差分超速离心，并通过0.22μm膜过滤器过滤。由于差示超速离心步骤的优化和过滤步骤的加入，该方法获得的sEV纯度高于经典差示超速离心。它为组织来源的sEV研究提供了重要的方法和支持数据。
Introduction
小细胞外囊泡（sEV）的直径约为30nm至150nm，由各种细胞分泌1。它们可以与组织细胞交流，通过将重要的生物分子（如脂质、蛋白质、DNA 和 RNA）运送到各种器官、组织、细胞和细胞内部分来调节局部或远处的微环境。因此，它们还可以改变受体细胞2，3的行为。特定sEV的分离和纯化是研究其在疾病发展和过程中的生物学行为的重要先决条件。差速超速离心 – 被视为金标准 – 通常用于将sEV与其通常所在的组织分离4。组织碎片、细胞碎片、大囊泡和凋亡小体可以通过这种技术去除，只留下sEV。
胶原酶D和DNase I已被证明不会影响细胞或囊泡的分子特征，这两种酶的特性都有助于细胞外基质中囊泡的释放5。这些酶已用于从人转移性黑色素瘤组织、结肠癌组织和结肠粘膜组织中提取 sEV5，6，7。然而，这些方法中胶原酶D和DNase I的浓度和消化时间不同，导致结论不一致。为了避免其他sEV亚型的共沉淀，研究人员通过过滤和/或差速离心去除了较大的细胞外囊泡（直径0.1μm或0.2μm8）。根据来源组织的不同，可能需要不同的分离和纯化方法9，10。
使用传统的差示超速离心方法从肝组织中提取sEV会在上清液表面形成一层白质，而无法确定其性质。在之前的研究11中，发现这层白质会影响sEV的纯度。虽然传统方法分离的样品颗粒数和蛋白质浓度高于现有方法，但变异系数较大，可能是因为许多污染物会导致结果的重复性差。也就是说，使用洗涤剂（即检测颗粒在1%Triton X-100中的溶解度），我们发现通过这种方法获得的sEV的纯度更高。因此，我们使用这种方法分离和纯化来自结直肠癌组织的sEV，用于蛋白质组学研究。
目前，sEVs在肝癌中的研究主要集中在血清、血浆和细胞培养物的上清液12，13，14上。然而，来源于肝癌组织的sEV可以更准确地反映肝癌的生理病理和周围微环境，有效避免其他EV的降解和污染15，16。利用差示超速离心，该方法可富产率，获得高质量的sEVs，为肝癌的进一步研究提供重要依据。该方法使肝癌组织能够通过尖锐分离分离，并通过胶原酶D和DNase I解离。然后，通过过滤和差示超速离心进一步去除细胞碎片、大囊泡和凋亡小体。最后，分离并纯化sEV以供后续研究。
Protocol
Representative Results
Discussion
该协议描述了从肝癌组织中提取sEV的可重复方法。通过尖锐的组织分离、消化酶处理、差示超速离心和 0.22 μm 滤膜过滤和纯化获得高质量的 sEV。对于下游分析，确保sEV的高纯度非常重要。在差速离心过程中，上清液表面会出现一层白色物质（成分不明）。由于该层会污染sEV，我们通过重复离心（3，000 × g 和12，000 × g）并通过0.22 μm膜过滤（补充表S1）尽可能去除它。由…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢赣南医科大学附属第一医院对这项工作的支持。这项工作得到了中国国家自然科学基金（批准号82260422）的支持。
Materials
| 0.22 µm Membrane Filter Unit | Millex | SLGPR33RB | |
| 1 mL Sterile syringe | Hubei Xianming Medical Instrument Company | YL01329 | |
| 2% Uranyl Acetate | Electron Microscopy Sciences | 22400-2 | |
| 4.7 mL Centrifuge Tube | Beckman Coulter | 361621 | |
| 6-well Cell cuture plate | LABSELECT | 11110 | |
| 50 mL Beaker | Tianjin Kangyiheng Experimental Instrument Sales Company | CF2100800 | |
| 70 µm Cell strainer | Biosharp | BS-70-XBS | |
| 100 mm Cell culture dish | CELL TER | CS016-0128 | |
| 600 µL Centrifuge tube | Axygen | MCT060C | |
| BCA protein quantification kit | Thermo Fisher | RJ240544 | |
| Beckman Coulter Optima-Max-TL | Beckman | A95761 | |
| BioRad Mini trans-blot | Bio-Rad | 1703930 | |
| BioRad Mini-Protean | Bio-Rad | 1645050 | |
| CD63 Antibody | Abcam | ab134045 | |
| CD9 Antibody | Abcam | ab263019 | |
| Centrifuge 5430R | Eppendorf | 5428HQ527333 | |
| Cleaning Solution | NanoFCM | C1801 | |
| Collagenase D | Roche | 11088866001 | |
| Copper net | Henan Zhongjingkeyi Technology Company | DJZCM-15-N1 | |
| Dry Thermostat | Hangzhou allsheng instruments company | AS-01030-00 | |
| FITC Anti Human CD9 Antibody | Elabscience | E-AB-F1086C | |
| Glycine | Solarbio | G8200 | |
| Goat horseradish peroxidase (HRP)-coupled secondary anti-mouse antibody | Proteintech | SA00001-1 | |
| Goat horseradish peroxidase (HRP)-coupled secondary anti-rabbit antibody | Proteintech | SA00001-2 | |
| Methanol | Shanghai Zhenxing Chemical Company | ||
| Nanoparticle flow cytometer | NanoFCM INC | FNAN30E20112368 | |
| Phosphatase inhibitors(PhosSTOP) | Roche | 4906845001 | |
| Phosphate Buffered Saline(PBS) | Servicebio | G4202 | |
| Polyvinylidene Difluoride Membrane | Solarbio | ISEQ00010 | |
| QC Beads | NanoFCM | QS2502 | |
| RPMI-1640 basic medium | Biological Industries | C11875500BT | |
| Scalpel | Guangzhou Kehua Trading Company | NN-0623-1 | |
| Silica Nanospheres | NanoFCM | S16M-Exo | |
| Transference Decoloring Shaker TS-8 | Kylin-Bell | E0018 | |
| Transmission Electron Microscope | Thermo Scientific | Talos L120C | |
| Tris | Solarbio | T8060 | |
| TSG101 Antibody | Proteintech | 28283-1-AP | |
| Tweezer | Guangzhou Lige Technology Company | LG01-105-4X |
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