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唾液细胞外囊泡的分离：基于超速离心方案的分析

Salivary Extracellular Vesicles Separation: Analysis of Ultracentrifugation-Based Protocols.

作者信息

Itzel Castillejos-García, Eduardo Martínez-Martínez, Velia Ramírez-Amador, Mireya Cisneros-Villanueva, Alfredo Hidalgo-Miranda, Pilar Ramos-Godínez María Del, Gabriela Anaya-Saavedra

机构信息

Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Ciudad de México, Mexico.

Laboratorio de Vesículas Extracelulares y Comunicación Celular, Instituto Nacional de Medicina Genómica, Ciudad de México, Mexico.

出版信息

Oral Dis. 2025 Mar;31(3):879-889. doi: 10.1111/odi.15171. Epub 2024 Oct 27.

DOI:10.1111/odi.15171
PMID:39462790
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12021316/
Abstract

INTRODUCTION

The clinical potential of extracellular vesicles (EVs) is widely acknowledged, yet the standardization and reproducibility of its separation remain challenging. This study compares three protocols: ultracentrifugation (UC), UC with purification step (UC + PS), and a combined protocol using polymer-based precipitation and UC (PBP + UC).

METHODS

Salivary samples were collected from healthy donors. EVs were separated (UC, UC + PS, and PBP + UC) and characterized using transmission electron microscopy, nanoparticle tracking analysis, EV purity, RNA concentration, and Western blotting. miRNA expression was evaluated by quantitative RT-PCR. Statistical analyses comparing groups were performed using ANOVA.

RESULTS

All methods successfully separated CD9+ and CD63+ EVs from saliva. The UC + PS and PBP + UC protocols yielded the highest concentrations of EVs, enriched in < 200 nm vesicles. EV purity and RNA recovery were comparable among all methods. Expression of miR-16, miR-27a, and miR-99a was successfully detected using all methods.

CONCLUSIONS

The UC + PS and PBP + UC protocols demonstrate comparable efficiency in separating salivary EVs. However, the combined PBP + UC protocol, with its simplified processing capability, offers a significant advantage, particularly in the initial phase of EV separation. This finding suggests its potential application in clinical settings where time-sensitive simple processing is critical. Further validation is needed to confirm its effectiveness for transcriptomic and proteomic analyses.

摘要

引言

细胞外囊泡（EVs）的临床潜力已得到广泛认可，但其分离的标准化和可重复性仍具有挑战性。本研究比较了三种方案：超速离心法（UC）、带有纯化步骤的超速离心法（UC + PS）以及使用基于聚合物的沉淀法和超速离心法的联合方案（PBP + UC）。

方法

从健康供体收集唾液样本。分离EVs（UC、UC + PS和PBP + UC），并使用透射电子显微镜、纳米颗粒跟踪分析、EV纯度、RNA浓度和蛋白质免疫印迹法对其进行表征。通过定量逆转录聚合酶链反应评估miRNA表达。使用方差分析对各组进行统计分析。

结果

所有方法均成功从唾液中分离出CD9 +和CD63 + EVs。UC + PS和PBP + UC方案产生的EVs浓度最高，富含<200 nm的囊泡。所有方法的EV纯度和RNA回收率相当。使用所有方法均成功检测到miR - 16、miR - 27a和miR - 99a的表达。

结论

UC + PS和PBP + UC方案在分离唾液EVs方面表现出相当的效率。然而，联合的PBP + UC方案具有简化的处理能力，具有显著优势，特别是在EV分离的初始阶段。这一发现表明其在对时间敏感的简单处理至关重要的临床环境中的潜在应用。需要进一步验证以确认其在转录组学和蛋白质组学分析中的有效性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb81/12021316/b0f51b62746b/ODI-31-879-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb81/12021316/9b448b0a9454/ODI-31-879-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb81/12021316/8167b7d018d9/ODI-31-879-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb81/12021316/390999cb145e/ODI-31-879-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb81/12021316/b0f51b62746b/ODI-31-879-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb81/12021316/9b448b0a9454/ODI-31-879-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb81/12021316/8167b7d018d9/ODI-31-879-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb81/12021316/390999cb145e/ODI-31-879-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb81/12021316/b0f51b62746b/ODI-31-879-g002.jpg

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Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.
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Do Media Extracellular Vesicles and Extracellular Vesicles Bound to the Extracellular Matrix Represent Distinct Types of Vesicles?媒体细胞外囊泡和与细胞外基质结合的细胞外囊泡是否代表不同类型的囊泡？
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