不同血清量标本外泌体提取及微小RNA的检测结果比较

本文旨在探讨Qiagen exoRNeasy Serum/Plasma试剂盒提取血清标本中外泌体所需的最适血清量。采用Qiagen exoRNeasy Serum/Plasma 试剂盒分别对250、500、1 000 μL血清中的外泌体进行抽提，使用透射电子显微镜检测分离的外泌体大小和形态，蛋白质免疫印迹法检测外泌体蛋白标记CD63和TSG101的表达，实时荧光定量聚合酶链反应(polymerase chain reaction，PCR)检测外泌体中微小RNA-122(microRNA-122，miR-122)的表达。结果显示，透射电子显微镜下可见血清外泌体呈圆形或椭圆形，直径30～150 nm，有完整的膜结构。蛋白免疫印迹法检测外泌体CD63和TSG101阳性。实时荧光定量PCR检测慢性乙型肝炎患者250、500、1 000 μL血清外泌体中miR-122表达量，与正常人相比，分别上调22.44、21.48、20.69倍(P＝0.42)。结果提示，在临床血清样本体积有限的情况下，采用 Qiagen exoRNeasy Serum/Plasma 试剂盒提取血清中外泌体，减少血清量至250 μL也可达到所需实验目的。

Comparison of exosomes and microRNAs isolated from different gradient serum samples

The present paper aims to investigate the optimum amount of serum samples used to isolate exosomes by Qiagen exoRNeasy Serum/Plasma Midi Kit. Exosomes were isolated by Qiagen exoRNeasy Serum/Plasma Midi Kit from different gradient serum samples, including 250 μL, 500 μL, 1 000 μL. The particle size and morphology of exosomes were measured by transmission electron microscopy. The expressions of exosomal surface protein markers CD63 and TSG101 were determined by Western blotting. The expression of microRNA-122 (miR-122) in exosomes was detected by real-time fluorescent quantitative polymerase chain reaction (PCR). Serum exosomes were circular or elliptic under the transmission electron microscope with diameters of 30-150 nm and they had intact membrane structure. Western blotting results showed that CD63 and TSG101 were positive in exosomes. The real-time fluorescent quantitative PCR results showed that when the initial serum volume was 250 μL, 500 μL and 1 000 μL, the expression of miR-122 in chronic hepatitis B patients was up-regulated by 22.44 folds, 21.48 folds and 20.69 folds, respectively compared with controls (P＝0.42). It is suggested that a small amount of 250 μL serum could be used for isolation of exosomes by Qiagen exoRNeasy Serum/Plasma Midi Kit, when the clinical serum sample is limited.