聚乙二醇沉淀剂有效分离尿外泌体(英文)  

Effective Isolation of Urinary Exosomes by Novel Approach: Polyethylene Glycol Precipitation

作　　者：吕春燕[1] 汪弋力[2] 丁维俊[2] 

机构地区：[1]成都中医药大学临床医学院病理科/病理教研室,成都610072 [2]成都中医药大学基础医学院基础医学实验中心,成都610072

出　　处：《中国生物化学与分子生物学报》2018年第1期110-116,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：成都中医药大学校基金(No.CGPY1601)和成都中医药大学实验技术项目(No.SYZD2017002)

摘　　要：尿外泌体是病毒大小的胞外囊泡,是非侵入性获得肾及泌尿生殖道细胞生理病理信息的重要靶标。聚乙二醇沉淀剂可经济高效地分离富集血清等外泌体,但未见用于尿外泌体富集的详细报道。本研究采用聚乙二醇沉淀剂分离鉴定尿外泌体,并对其RNA组分进行检测,以期建立一个经济、高效、简便的尿外泌体分离富集方法。采集10例健康志愿者晨尿20 m L,聚乙二醇沉淀剂分离尿外泌体。透射电镜观察到直径30~100 nm双层膜包绕的囊性小泡,中央有直径5~15 nm高电子密度区。Western印迹检测到外泌体标记蛋白CD63、CD9、TSG101、ADAM10和内标蛋白β-肌动蛋白的表达。纳米粒径仪测定粒子直径介于30~130 nm,并可见25.37 nm和95.07 nm二个粒子峰。qRT-PCR扩增得到β-肌动蛋白和RNU6 RNA产物带。上述结果表明,聚乙二醇沉淀剂可分离富集尿外泌体,该法简单、高效,不需要超速离心机等昂贵设备,且采用该法富集到的外泌体可用于后续蛋白质与核酸分析。该方法可望加速液体活检应用,尤其是肾及泌尿生殖道病变的无创检测。Urinary exosomes are virus-sized vesicles or sacs including the physiological and pathological information originated from the kidney and urogenital tracts. The approach of polyethylene glycol precipitation （PGP） has been successfully and effectively utilized in the isolation and enrichment of exosomes from serum samples. However, there is a shortage of detailed reports focused on the isolation of urinary exosomes by PGP. In this study, we isolated and enriched the urinary exosomes by PGP. Urine samples of ten healthy volunteers were recruited. Results of electron microscope observation displayed the cystoid vesicles wrapped in double layer membranes in diameters from 30 to 100 nm, and with a high electron density area. CD63, CD9, TSG101 and ADAM10 as the biomarkers of urinary exosomes were detected by Western blot analysis. Two peaks, at 25.37 and 95.07 nm respectively, were observed at all isolated urinary exosomes. Representative exosomal RNAs （SnRNA-U6 and β-actin） were detected by qRT-PCR in all samples. Our results suggest that PGP method is an effective, economical and convenient way for the isolation of urinary exosomes, which might accelerate the application of noninvasive liquid biopsy, especially suitable for kidney and urogenital tract diseases.

关 键 词：尿外泌体 聚乙二醇沉淀法 RNA检测 

分 类 号：R-331[医药卫生]