一种简单、经济、高产的聚乙二醇提取卵泡和血清来源的细胞外囊泡的方法。

IF 1.4 Q2 MEDICINE, GENERAL & INTERNAL
Tzu Chi Medical Journal Pub Date : 2024-10-25 eCollection Date: 2025-01-01 DOI:10.4103/tcmj.tcmj_160_24
Aye Aye Khine, Pao-Chu Chen, Ying-Hsi Chen, Sung-Chao Chu, Yu-Shuan Chen, Hsuan-Shun Huang, Tang-Yuan Chu
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引用次数: 0

摘要

目的:研究聚乙二醇(PEG)基细胞外囊泡(ev)从人卵泡液(FF)和血清中提取的最佳工艺条件,并对其进行功能分析。将基于peg的EV结果与基于ExoQuick (ExoQ)的EV结果进行比较。材料和方法:采用不同浓度PEG(8000)提取ff - ev和血清ev。采用纳米颗粒跟踪分析对颗粒进行计数,并对电动汽车进行电子显微镜观察。外泌体通过外泌体特异性标记物的western blot分析证实。从外泌体中提取RNA和microRNA,进行定量聚合酶链反应分析。利用输卵管上皮细胞(FTE)进行EV摄取实验和非锚定生长实验,以证实提取的EV具有转化活性。结果:PEG 8%富集法产率最高,携带蛋白最低。含PEG 8%的盐比不含PEG 8%的盐产量高。从FF中提取PEG 8%和exoq为基础的EV,隔夜富集量分别增加了4倍和18倍。对于血清EV,同样的隔夜富集可适度提高PEG法和ExoQ法的产量(8%)。携带蛋白的减少导致ev促进的转化活性增加。结论:本研究克服了耗时、昂贵、费力、复杂的机器依赖EV提取方法。研究表明,从FF中提取EV需要较长的孵育时间。与exoq提取相比,PEG 8000提取的EV提取率更高,携带的蛋白质更少。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A simple, economical, and high-yield method for polyethylene glycol-based extraction of follicular and serum-derived extracellular vesicles.

Objectives: The optimization of polyethylene glycol (PEG)-based extracellular vesicles (EVs) extraction from human follicular fluid (FF) and serum was investigated, and their functional analysis was confirmed. The PEG-based EV results were compared to the ExoQuick (ExoQ)-based EV.

Materials and methods: FF-EVs and serum-EVs were extracted by using different concentrations of PEG (8000). Nanoparticle tracking analysis was used to count the particles, and electron microscopy of EVs was performed for visualization. Exosomes were confirmed by the western blot analysis with exosome-specific markers. RNA and microRNA were extracted from exosomes and quantitative polymerase chain reaction analysis was performed. Fallopian tube epithelial (FTE) cells were used for the EV uptake experiment and an anchorage-independent growth test to confirm that extracted EVs harbor transformation activity.

Results: The PEG 8% enriched method produced the highest yield and the lowest carry-over protein. Salt containing PEG 8% produced a higher yield than nonsalted PEG 8%. Overnight enrichment increased four times and 18 times for PEG 8% and ExoQ-based EV extraction from FF. For serum EV, the same overnight enrichment moderately increased yield for both PEG 8% and ExoQ methods. Less carry-over protein resulted in more EV-promoted transformation activity.

Conclusion: This study overcomes the time-consuming, expensive, laborious, and complicated machine-dependent EV extraction methods. The study highlights that longer incubation time is needed for EV extraction from FF. PEG 8000-based EV extraction provided a higher yield and less carry-over protein than ExoQ-based EV extraction.

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来源期刊
Tzu Chi Medical Journal
Tzu Chi Medical Journal MEDICINE, GENERAL & INTERNAL-
CiteScore
3.40
自引率
0.00%
发文量
44
审稿时长
13 weeks
期刊介绍: The Tzu Chi Medical Journal is the peer-reviewed publication of the Buddhist Compassion Relief Tzu Chi Foundation, and includes original research papers on clinical medicine and basic science, case reports, clinical pathological pages, and review articles.
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