a Laboratory of Food and Environmental Hygiene, Cooperative Department of Veterinary Medicine , Gifu University , Gifu, Japan.
b The United Graduate School of Veterinary Sciences , Gifu University , Gifu, Japan.
Drug Dev Ind Pharm. 2019 Mar;45(3):359-364. doi: 10.1080/03639045.2018.1539743. Epub 2018 Nov 7.
This study aimed to establish a rapid and simple method for isolating exosomes from raw bovine milk and to compare the quality of the isolated exosomes with those isolated by a standard method involving ultracentrifugation (UC).
To remove caseins, which are major milk proteins consisting more than 80% of milk protein (35% in human breast milk) and hamper isolation and purification of exosomes, hydrochloride (HCl) was added to milk for isoelectric precipitation (IP). The effects of acidification on morphological features, particle size distribution, surface charge, and exosome surface proteins were analyzed by electron microscopy, tunable resistive pulse sensing (TRPS), and Western blot (WB) analysis, respectively.
Electron microscopy showed that some of the exosomes isolated using IP had rough surfaces; most exosomes were successfully isolated without breakage, and their morphological features were similar to those of exosomes isolated by UC. TRPS showed that their surface charge and peaks (mode) for particle size distribution did not significantly differ between both methods. WB analysis using antibodies against the exosome surface marker proteins - milk fat globule-epidermal growth factor 8 (MFG-E8) and CD63 - revealed that the structures of exosome surface proteins were not affected by adding HCl.
IP can be used to remove caseins to reduce operation time. This method will be useful for efficient isolation and purification of bovine milk exosomes and contribute to progression of research on health management of dairy cattle and drug delivery systems in human medicine, which require large amounts of milk exosomes.
本研究旨在建立一种从生牛乳中快速简便分离外泌体的方法，并比较该方法与传统超速离心（UC）法分离外泌体的质量。
为了去除乳清蛋白（乳清蛋白占牛奶蛋白的 80%以上（人乳中占 35%），会阻碍外泌体的分离和纯化），向牛奶中添加盐酸（HCl）进行等电沉淀（IP）。通过电子显微镜、可调电阻脉冲传感（TRPS）和 Western blot（WB）分析分别研究酸化对形态特征、粒径分布、表面电荷和外泌体表面蛋白的影响。
电子显微镜显示，使用 IP 分离的一些外泌体表面粗糙；大多数外泌体在不破乳的情况下成功分离，其形态特征与 UC 分离的外泌体相似。TRPS 显示，两种方法的表面电荷和粒径分布峰值（模式）没有显著差异。使用针对外泌体表面标记蛋白 - 乳脂肪球表皮生长因子 8（MFG-E8）和 CD63 - 的抗体进行 WB 分析表明，HCl 的添加并未影响外泌体表面蛋白的结构。
IP 可用于去除乳清蛋白以缩短操作时间。该方法将有助于高效分离和纯化牛乳外泌体，并为奶牛健康管理和人用药物传递系统的研究提供帮助，这些研究需要大量的牛乳外泌体。
