超速离心法与聚乙二醇沉淀法提取人多发性骨髓瘤细胞来源外泌体的比较  

Comparison of Ultracentrifugation and PEG Precipitation Method for Extraction of Human Multiple Myeloma Cell-Derived Exosomes

作　　者：熊文杰[1] 刘焕勋[1] 史敦云[1] 楼瑾[1] 张琼丽[1] XIONG Wen-jie, LIU Huan-xun, SHI Dun-yun, LOU Jin, ZHANG Qiong-li(Shenzhen Second People＇s Hospital, Guangdong Shenzhen 51803)

机构地区：[1]深圳市第二人民医院,广东深圳518035

出　　处：《深圳中西医结合杂志》2018年第2期6-8,共3页Shenzhen Journal of Integrated Traditional Chinese and Western Medicine

基　　金：深圳市科技计划项目资助课题(JCYJ20150330102401094)

摘　　要：目的:鉴定比较超速离心法和聚乙二醇(PEG)沉淀法提取人多发性骨髓瘤细胞来源外泌体。方法:人多发性骨髓瘤细胞系RPMI 8226经去除外泌体无血清培养24~48 h培养后,分别使用超速离心法和PEG沉淀法进行外泌体的提取,分别测定两种方法所得外泌体的蛋白含量,并进行电镜鉴定比较。结果:超速离心法所提取外泌体测得实际浓度为(1.095±0.031)μg·μL^(-1),沉淀法所提取外泌体实际浓度为(2.069±0.086)μg·μL^(-1),组间比较,差异具有统计学意义(P<0.001);超速离心法测得外泌体蛋白总量(109.513±3.113)μg,沉淀法所提取外泌体测得蛋白总量(206.883±8.580)μg,组间比较,差异具有统计学意义(P<0.001)。电镜下超速离心法分离外泌体清晰可见,沉淀法提取的外泌体含有较多杂质,影响电镜观察。结论:超速离心法方法简单,耗时少,外泌体纯度高,可用于外泌体的蛋白、DNA和免疫功能研究。沉淀法耗时较长,所得外泌体杂质蛋白含量多,不适用于外泌体蛋白分析,可用于RNA分析。Objective To identify and compare human multiple myeloma cell-derived exosomes isolated by the ways of ultracentrifugation and polyethylene glycol precipitation. Methods Human myeloma cell line RPMI 8226 was cultured for 24-48h in serum-free medium removal of exosomes. Exosomes was isolated separately by the ways of ultracentrifugation and polyethylene glycol precipitation. Then determined separately protein content of the exosomes isolated by two methods, identified and compared them by electron microscope. Results The actual concentration of exosome extracted by the way of ultracentrifugation was 1.095 4- 0.031 μg · μL-1, whereas by precipitation was 2.069 ± 0.086μg · μL-1, the difference was statistically significant （P 〈 0.001）; The protein content of exosome by ultracentrifugation was 109.513 ± 3.113 g, whereas by precipitation was 109.513 ± 8.580 g. The difference was statistically significant （P 〈 0.001）. Under electron microscope, the exosome isolated by ultracentrifugation was clearly visible, and by precipitation method contained more impurities, which disturbed the observation under electron microscope. Conclusion The method of ultracentrifugation is simpler, shorter of time consuming than the method of polyethylene glycol precipitation. The exosome by ultracentrifugation is higher purity. It can be used in the study of protein, DNA and immune function of exosome. The precipitation method takes longer time. The exosome isolated by it contain more protein, and is not suitable for the analysis of exosome protein. It can be used in RNA analysis.

关 键 词：多发性骨髓瘤 外泌体 超速离心法 聚乙二醇沉淀法 

分 类 号：R730[医药卫生—肿瘤]