高效提取细胞培养上清外泌体方法的建立  

Development of a new method for efficiently isolating exosomes from cell culture supernatant

作　　者：王小鑫 王丽丽 张益国 高姣 马磊[1] 张婷婷[1] 陈国柱 WANG Xiaoxin;WANG Lili;ZHANG Yiguo;GAO Jiao;MA Lei;ZHANG Tingting;CHEN Guozhu(College of Life Sciences,Shihezi University,Shihezi 832000,Xinjiang UygurAutonomous Region,China)

机构地区：[1]石河子大学生命科学学院,新疆维吾尔自治区石河子832000 [2]京奥秘佳得医药科技有限公司,北京102600

出　　处：《中国生物制品学杂志》2024年第8期975-982,988,共9页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31860308);石河子大学高层次人才启动项目(RCZK201953)。

摘　　要：目的建立一种快速、简单且高效提取细胞培养上清中外泌体的方法,以期促进外泌体在临床与疾病治疗中的应用。方法将羟基(OH)修饰磁珠与高分子化合物Buffer EXD结合,建立新的外泌体提取系统,并使用该系统提取细胞培养上清中的外泌体。通过Western blot法检测外泌体标志蛋白的表达变化;纳米颗粒追踪分析(nanoparticles tracking analysis,NTA)技术检测外泌体的粒径大小和浓度;透射电子显微镜(transmission electron microscope,TEM)技术检测外泌体的形态结构。对Buffer EXD系统提取外泌体的方法进行优化(提取pH、Buffer EXD比例、孵育时间、磁珠用量及种类)。结合细胞培养和显微拍照技术分析新系统纯化的外泌体对A549细胞增殖的影响。结果新系统提取的外泌体样品中标志蛋白Alix、CD9和CD81等表达水平较高,而外泌体阴性蛋白Calnexin表达较少;新系统提取的外泌体粒径峰值在100 nm左右,具有茶托状的典型形状。新系统的pH为5.0,Buffer EXD浓度为20%,1.75%的OH磁珠与样品孵育时间为40 min时,提取的外泌体样品中标志蛋白含量最高。新系统提取的外泌体对A549细胞的增殖具有明显的促进作用。结论以OH修饰的磁珠为介质,结合高分子化合物Buffer EXD,成功建立了一种可快速、简单、高效地从细胞上清中提取结构完整、形态特征明显且具有生物学功能外泌体的新体系。Objective To develop a rapid,simple and efficient method for isolating exosomes from cell culture supernatant,so as to promote the application of exosomes in clinical and disease treatment.Methods A new exosomes extraction system was developed by combining hydroxyl(OH)modified magnetic beads with the polymeric compound Buffer EXD,and the exosomes were extracted from cell culture supernatant by using this system.The expression of exosome marker proteins was detected by Western blot,the particle size and concentration of exosomes were measured by nanoparticles tracking analysis(NTA),and the morphology and structure were determined by transmission electron microscope(TEM).In addition,the extraction methods of exosomes by Buffer EXD system were optimized(pH,Buffer EXD ratio,incubation time,magnetic bead dosage and type),and the effect of exosomes purified by the new system on the proliferation of A549 cells was analyzed by combining cell culture with photomicrograph.Results In exosomes purified by the new system,the expression levels of marker proteins Alix,CD9 and CD81 were high,while the expression level of exosome negative protein Calnexin was low.The exosomes extracted by the new system had a particle size peak of around 100 nm and a classical saucer-like shape.When the new system pH was 5.0,Buffer EXD concentration was 20%,and 1.75%OH magnetic beads were incu-bated with samples for 40 min,the content of marker proteins in exosomes was the highest.Additionally,the exosomes puri-fied by the new system significantly facilitated the proliferation of A549 cells.Conclusion Using OH-modified magnetic beads as the medium,combined with polymeric compound Buffer EXD,a new system was successfully established,which can isolate exosomes with complete structure,obvious morphological characteristics and biological functions from cell culture supernatant rapidly,simply and efficiently.

关 键 词：外泌体 细胞培养上清 高分子化合物Buffer EXD 磁珠 

分 类 号：Q819[生物学—生物工程]