Bok Eun-Yeong, Seo Sang Yeong, Lee Han Gyu, Wimalasena Sudu Hakuruge Madusha Pramud, Kim Eunju, Cho Ara, Jung Young-Hun, Hur Tai-Young, So Kyoung-Min, Lee Sung-Lim, Do Yoon Jung
Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea.
College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea.
J Anim Sci Technol. 2024 Sep;66(5):1021-1033. doi: 10.5187/jast.2024.e45. Epub 2024 Sep 30.
Exosomes have been extensively studied as disease biomarker in humans, given their role in transporting bioactive molecules. However, despite the great potential of exosomes as noninvasive diagnostic markers and therapeutic nanocarriers for bovine diseases, few studies have been conducted on bovine exosome. Thus, this study aimed to quantitatively and qualitatively compare three isolation methods to identify a suitable method for bovine serum. Exosomes were isolated using ultracentrifugation alone (UC), a combination of ultracentrifugation and size exclusion chromatography (US), or membrane affinity-based exoEasy kit (EE). Isolated particles were evaluated using a range of complementary techniques. Transmission electron microscopy showed that all three isolation methods resulted in particles with a cup-shaped morphology. The particle concentration measured by nanoparticle trafficking analyzer of US was lower compared to those of UC and EE method. As a result of immunoblotting, exosome markers including TSG101, CD81, and HSP70 were detected in US particles, while in UC and EE, only TSG101 expression was confirmed. Particles isolated from UC and EE showed a contamination with the blood protein albumin, whereas particles from US did not show albumin contamination. In addition, to evaluate the possibility of using exosomes as biomarkers, the profiles of the small RNA in the exosomes were compared using the bioanalyzer 2100. As a result, in the EE method, the band of small RNA (25-200 nt) was most prominent, and in the US methods, a distinct band was observed in the small RNA range. Collectively, the purity of exosomes without non-exosomal contamination was highest in the US method. However, for the detection of small RNA, the EE method was found to be the most suitable. Therefore, the results suggest that the optimal isolation method varies depending on the specific purpose of exosome isolation.
鉴于外泌体在运输生物活性分子方面的作用，其已作为人类疾病生物标志物得到广泛研究。然而，尽管外泌体作为牛病的非侵入性诊断标志物和治疗性纳米载体具有巨大潜力，但针对牛外泌体的研究却很少。因此，本研究旨在对三种分离方法进行定量和定性比较，以确定一种适用于牛血清的方法。外泌体通过单独使用超速离心法（UC）、超速离心与尺寸排阻色谱法相结合（US）或基于膜亲和的外泌体提取试剂盒（EE）进行分离。使用一系列互补技术对分离出的颗粒进行评估。透射电子显微镜显示，所有三种分离方法均产生了杯状形态的颗粒。与UC和EE方法相比，US方法通过纳米颗粒运输分析仪测得的颗粒浓度较低。免疫印迹结果显示，在US分离的颗粒中检测到了包括TSG101、CD81和HSP70在内的外泌体标志物，而在UC和EE分离的颗粒中，仅证实了TSG101的表达。从UC和EE分离的颗粒显示有血液蛋白白蛋白污染，而US分离的颗粒未显示白蛋白污染。此外，为了评估使用外泌体作为生物标志物的可能性，使用生物分析仪2100比较了外泌体中小RNA的谱图。结果，在EE方法中，小RNA（25 - 200 nt）条带最为明显，而在US方法中，在小RNA范围内观察到一条明显的条带。总体而言，US方法中外泌体的纯度最高，不存在非外泌体污染。然而，对于小RNA的检测，发现EE方法最为合适。因此，结果表明最佳分离方法因外泌体分离的具体目的而异。
