不同保存时间的血浆及血清中外泌体生物学特性的对比  

Biological features of exosomes from the plasma and serum preserved for different periods of time

作　　者：王婷婷[1] 韩影 高芳芳[2] 叶磊[2] 张育军 WANG Ting-ting;HAN Ying;GAO Fang-fang;YE Lei;ZHANG Yu-jun(Department of Gastroenterology,Peking University People’s Hospital,Beijing 100044,China;The Central Lab,Peking University People’s Hospital,Beijing 100044,China)

机构地区：[1]北京大学人民医院消化内科,北京100044 [2]北京大学人民医院中心实验室,北京100044

出　　处：《医学研究生学报》2019年第7期705-709,共5页Journal of Medical Postgraduates

基　　金：北京大学人民医院研究与发展基金(RDX2018-05)

摘　　要：目的外泌体对于疾病的诊断和治疗具有不可比拟的优势,但其保存方法、保存时间、保存效果的评估研究较少。文章旨在观察不同保存时间的血浆及血清中外泌体的大小、密度、形态及阳性蛋白表达有无改变。方法使用Exoquick试剂盒分别提取新鲜及保存在-80℃冰箱1年、3年、5年的正常人血浆及血清中的外泌体;使用纳米粒度颗粒跟踪分析仪对提取的外泌体进行粒径分析;使用透射电镜对提取的外泌体的形态进行观察;Western blot检测外泌体标志物TSG101和CD63的表达改变。结果保存时间达3年的血浆中的外泌体粒径较大(30~200nm),血清较血浆中外泌体数量略高;电镜下不同保存时间的血浆及血清中外泌体形态变化不明显;随着保存时间的延长,血浆及血清中外泌体蛋白TSG101及CD63含量发生降低,其中CD63降低尤其明显。结论不同保存时间的血浆及血清中外泌体粒径及形态变化差异不大,但其内含蛋白标志物随着时间延长发生明显减少,因此进行外泌体蛋白的相关研究时推荐新鲜提取立即实验。Objective Exosomes play an extremely important role in the diagnosis and treatment of diseases, but very few studies have been reported on the method, time and results of their preservation. We observed the changes in the size, density, morphology and protein expression of exosomes in the plasma and serum preserved for different lengths of time. Methods Using the Exoquick kit, we isolated exosomes in the plasma and serum from healthy humans which were fresh or preserved at a temperature of -80℃ for 1, 3 or 5 years. We analyzed the numbers and diameters of the isolated exosomes with the nanoparticle-tracking analyzer NanoSight NS 300, observed their morphology under the transmission electron microscope, and detected the expressions of the exosome biomarkers TSG101 and CD63 by Western blot. Results The exosomes preserved for different periods of time ranged in diameter from 30 to 200 nm, more observed in the serum than in the plasma, and with no statistically significant difference in the morphological changes among different groups of preservation time. The expressions of the TSG101 and CD63 proteins in the exosomes were decreased with the prolonging of the preservation time, the latter even more significantly than the former. Conclusion There are no statistically significant differences in the diameter or morphology of the exosomes in the human plasma and serum preserved for different periods of time, but the protein biomarkers of the exosomes are decreased significantly with the prolonged time of preservation. Therefore, freshly isolated exosomes are recommended for experiments in the studies of exosome-related proteins.

关 键 词：外泌体 血清 血浆 保存 

分 类 号：R55[医药卫生—血液循环系统疾病]