Isolation of extracellular vesicles from intestinal tissue in a mouse model of intestinal ischemia/reperfusion injury
Isolation of extracellular vesicles from intestinal tissue in a mouse model of intestinal ischemia/reperfusion injury
复制标题
肠道缺血/再灌注损伤小鼠模型肠道组织中细胞外囊泡的分离
DOI：
10.2144/btn-2019-0159
复制
发表时间：
2020-05-01
期刊：
影响因子：
2.7
通讯作者：
Liu, Ke-Xuan
中科院分区：
文献类型：
--
作者：
Chen, Xiao-Dong;Zhao, Jin;Liu, Ke-Xuan
细胞外小泡(EV)是一种小的膜性颗粒，有助于细胞间的通讯。将电动汽车与组织分离仍然是一项技术挑战。在这里，我们提出了一种严格的方法，从小鼠肠缺血/再灌注(I/R)模型的肠道组织中提取EV，并分析它们的miRNA含量。经电子显微镜和NTA分析证实，分离的EVS呈典型的杯状，大小峰直径为120-130 nm。它们还表达EV标志物CD9、CD63、CD81、Tsg101和Alix。实时荧光定量聚合酶链式反应证实这些颗粒含有与I/R损伤相关的miRNAs。本研究提供了一种从肠道组织中分离肠道病毒的实用方法，该方法适用于miRNA分析等后续应用，为研究肠道I/R损伤机制提供了一种新的方法。该方法适用于miRNA分析等下游研究。
Extracellular vesicles (EVs) are small membranous particles that contribute to intercellular communications. Separating EVs from tissue is still a technical challenge. Here, we present a rigorous method for extracting EVs from intestinal tissue in a mouse intestinal ischemia/reperfusion (I/R) model, and for analyzing their miRNA content. The isolated EVs show a typical cup shape with a size peak of 120-130 nm in diameter, confirmed by TEM and NTA. They also express EV markers such as CD9, CD63, CD81, Tsg101 and Alix. Real-time qPCR confirmed that these pellets contain miRNAs related to I/R injury. Our study presents a practical way to isolate EVs from intestinal tissue which is suitable for downstream applications such as miRNA analysis, and provides a novel method for investigating the mechanism of intestinal I/R injury.METHOD SUMMARY We present a practical way to isolate EVs from intestinal tissue; the method consists of enzymatic digestion, differential centrifugation and density gradient centrifugation. This method is suitable for downstream studies such as miRNA analysis.