Vaswani Kanchan, Koh Yong Qin, Almughlliq Fatema B, Peiris Hassendrini N, Mitchell Murray D
University of Queensland Centre for Clinical Research, The University of Queensland, Brisbane, Queensland, 4029, Australia.
University of Queensland Centre for Clinical Research, The University of Queensland, Brisbane, Queensland, 4029, Australia.
Reprod Biol. 2017 Dec;17(4):341-348. doi: 10.1016/j.repbio.2017.09.007. Epub 2017 Oct 10.
Exosomes are nanovesicles that play important roles in intercellular communication as they carry information to target cells. Isolation of high purity exosomes will aid in studying the exosomal cargo and quantity as well as how cell-specific messages are carried. We describe a new method incorporating size exclusion chromatography (SEC) to enrich milk-derived exosomes from extracellular vesicles (EVs). This involved the initial isolation of EVs from bovine milk via milk processing and ultracentrifugation; followed by a new method to enrich exosomes using SEC. This method was compared to buoyant density gradient centrifugation, a widely used method of enrichment. Exosomes were characterised by particle concentration and size (nanoparticle tracking analysis, NTA), morphology (transmission electron microscopy, TEM), presence of exosomal markers (immunoblotting) and protein concentration (bicinchoninic acid assay, BCA). Proteomic profiles of exosomal fractions were analyzed by mass spectrometry using Information Dependant Acquisition. Milk exosomal fractions were shown to contain exosomal markers flotillin-1 (FLOT-1) and tumor susceptibility gene-101 (TSG-101). The new method produced a higher yield of exosomes compared to buoyant density gradient centrifugation. Pooled exosomal fractions exhibited intact morphology by TEM. The use of SEC confirmed the fractionation of exosomes based on size while minimizing the interference with proteins. Tetraspanins CD9 and CD81 were observed via mass spectrometry in exosomal fractions. This new and efficient method confirmed the signatures for exosomes derived from unpasteurized bovine milk. Purification of exosomes is a foundational technique in the study of biomarkers for pathological conditions and effective drug delivery systems.
外泌体是纳米囊泡，在细胞间通讯中发挥着重要作用，因为它们能将信息传递给靶细胞。分离高纯度外泌体将有助于研究外泌体的货物、数量以及细胞特异性信息的传递方式。我们描述了一种结合尺寸排阻色谱法（SEC）从细胞外囊泡（EVs）中富集牛奶来源外泌体的新方法。这包括首先通过牛奶加工和超速离心从牛乳中分离EVs；然后采用一种利用SEC富集外泌体的新方法。该方法与广泛使用的浮力密度梯度离心法进行了比较。通过颗粒浓度和大小（纳米颗粒跟踪分析，NTA）、形态（透射电子显微镜，TEM）、外泌体标志物的存在（免疫印迹）和蛋白质浓度（二辛可宁酸测定，BCA）对外泌体进行了表征。使用信息依赖采集的质谱法分析了外泌体组分的蛋白质组学图谱。牛奶外泌体组分显示含有外泌体标志物 flotillin-1（FLOT-1）和肿瘤易感基因-101（TSG-101）。与浮力密度梯度离心法相比，新方法产生的外泌体产量更高。通过TEM观察，合并的外泌体组分呈现完整的形态。SEC的使用证实了基于大小对外泌体进行分级分离，同时将对蛋白质的干扰降至最低。通过质谱法在外泌体组分中观察到了四跨膜蛋白CD9和CD81。这种新的高效方法确定了来自未巴氏杀菌牛乳的外泌体的特征。外泌体的纯化是研究病理状况生物标志物和有效药物递送系统的一项基础技术。
