CHO-K1细胞培养上清及其外泌体的蛋白质组分析  

Proteomic Analysis of CHO-K1 Cell Culture Supernatant and Exosomes

作　　者：李浪 吴桐宇 姚宇峰 黄青丽 严继舟 LI Lang;WU Tongyu;YAO Yufeng;HUANG Qingli;YAN Jizhou(School of Resources Environmental&Chemical Engineering,Na nchang University,Jiangxi Nanchang 330031;Cellgene Bioscience,Shanghai 200120)

机构地区：[1]南昌大学资源环境与化工学院,江西南昌330031 [2]上海赛唐生物技术有限公司,上海201308

出　　处：《生物化工》2021年第1期63-69,共7页Biological Chemical Engineering

摘　　要：目的:确定CHO-K1细胞能否够分泌外泌体,运用液相色谱-质谱联用技术鉴定无血清培养条件下CHO-K1细胞培养上清中的蛋白质和外泌体中蛋白质的成分。方法:将CHO-K1细胞驯化至无血清培养基中,采用exoRNeasy血清/血浆试剂盒提取培养基中的外泌体,透射电镜复染法鉴定其大小和形态,质谱法鉴定标志蛋白;提取培养上清和外泌体中的蛋白,运用nanoLC-ESI-MS-MS技术进行鉴定,对鉴定到的蛋白质进行基因本体论(GO)注释分析。结果:从培养上清样本中鉴定到1243种蛋白质,分子质量范围在7~559 kDa,等电点(PI)在3~11;透射电镜下可见外泌体的双层膜小囊泡结构;从外泌体样本中鉴定到1259种蛋白质,分子质量范围在5~4007 kDa,等电点在3~12,其中鉴定到外泌体标志蛋白有膜连蛋白、肿瘤易感蛋白、Rab蛋白、热休克蛋白-90和热休克蛋白-60;GO注释结果显示上清蛋白和外泌体蛋白主要都来源于细胞器和细胞的膜结构。结论:形态学和标志蛋白的鉴定结果确认CHO-K1细胞的培养上清中存在外泌体,并初步构建了无血清条件下CHO-K1细胞的培养上清蛋白质和外泌体蛋白质的质谱数据集。Objective:To confirm that CHO-K1 cells can secrete exosomes,using LC-MS to identify protein components of CHO-K1 cell culture supernatant under serum-free culture conditions and the protein components in the exosomes.Methods:Adopting the serum-free medium domestication cell,exosomes were isolated by exoRNeasy Serum/Plasma Starter Kit from the culture medium.The particle size and morphology of exosomes were measured by transmission electron microscopy.The expressions of exosomal surface protein markers were determined by mass spectroscopy.Using nanoLC-ESI-MS-MS technology to identify proteins in culture supernatant and exosomes,perform gene ontology annotation analysis on the identified proteins.Results:1243 proteins were identified from the culture supernatant samples,the molecular masses range from 7~559 kDa,and the isoelectric point(PI)is between 3~11.The small vesicle structure of the double membrane of exosomes under the transmission electron microscope 1259 proteins were identified from exosomal samples with molecular masses ranging from 5~4007 kDa and isoelectric points between 3~12.And exosomal marker proteins were identified:annexin,tumor-susceptible protein,Rab protein,heat shock protein-90,heat shock protein-60.The annotation results of gene ontology show that cell culture supernatant proteins and exosomal proteins are mainly derived from organelles and membrane structures.Conclusion:The identification results of morphology and marker protein confirmed the presence of exosomes in the culture supernatant of CHO-K1 cells.And preliminarily constructed a mass spectrum data set of CHO-K1 cell culture supernatant protein and exosomal protein under serum-free conditions.

关 键 词：CHO-K1细胞系 上清蛋白 外泌体 蛋白质组 质谱 

分 类 号：Q26[生物学—细胞生物学]