利用定量蛋白组学的方法研究甲异靛诱导K562细胞凋亡的机制  

Exploration of Mechanism for Meisoindigo-Inducing K562 Cell Apoptosis by Using Quantitative Proteomic Analysis

作　　者：毛新荷 徐颖茜 邢海燕 田征 唐克晶 刘璐 饶青 王敏 王建祥 MAO Xin-He;XU Ying-Xi;XING Hai-Yan;TIAN Zheng;TANG Ke-Jing;LIU Lu;RAO Qing;WANG Min;WANG Jian-Xiang(State Key Laboratory of Experimental Hematology,Institute of Hematology &Blood Disease Hospital,Chinese Academy of Medical Sciences &Peking Union Medical College,Tianjin 300020,China;Jingjie PTM BioLab Co Ltd,Hangzhou Economic &Technologic Development Area,Hangzhou 310018,Zhejiang Province,China)

机构地区：[1]中国医学科学院北京协和医学院血液病医院(血液学研究所)实验血液学国家重点实验室,天津300020 [2]杭州景杰生物科技有限公司,浙江杭州310081

出　　处：《中国实验血液学杂志》2018年第6期1589-1597,共9页Journal of Experimental Hematology

基　　金：天津市临床医学研究中心建设项目(15ZXLCSY00010);实验血液学国家重点实验室自主研究课题(Z17-04)

摘　　要：目的:利用串联质谱标签系统(tandem mass tag,TMT)标记定量蛋白组学的方法,检测白血病细胞系K562细胞经甲异靛(meisoindigo)处理后蛋白表达的改变,探讨甲异靛诱导白血病细胞凋亡的作用机制。方法:用CCK8法检测甲异靛对K562细胞的半抑制浓度(inhibitory concentration 50,IC50),流式细胞术检测不同浓度甲异靛诱导K562细胞的凋亡水平。K562细胞经终浓度为0. 2%DMSO(对照组)和20μmol/L甲异靛(实验组)处理2 h后,提取总蛋白,TMT标记,高效液相色谱分级和质谱定量蛋白组学技术鉴定肽段和丰度信息,并进行3次技术重复。用Mascot软件鉴定蛋白,用GO(gene ontology)注释、富集和聚类分析方法分析差异表达蛋白。结果:在K562细胞系中,甲异靛以剂量依赖的方式诱导K562细胞凋亡(r=0. 98);蛋白质组学分析结果显示,共鉴定出蛋白质5 544个,其中有定量数据的蛋白质4 792个。与对照组相比,在实验组中表达差异1. 5倍以上的蛋白有8个,其中4个蛋白表达上调,4个蛋白表达下调,差异变化的蛋白主要与活性氧代谢相关。结论:应用定量蛋白质组学分析表明,包括DDIT4等蛋白的表达在甲异靛处理K562细胞系早期发生明显改变,提示活性氧代谢过程的改变可能在甲异靛促进凋亡的机制中发挥了重要作用。Objective: To screen the differentially expressed proteins at the early stage of K562 cells treated with meisoindigo by using tandem mass tags ( TMT ) -based proteomics technology,and to explore the mechanism for meisoindigo - inducing apoptosis.Methods: The half inhibitory concentration ( IC50 ) of mesoindigo on K562 cells was determined by CCK8.The flow cytometry was used to assay the apoptosis of K562 cells treated by meisoindigo or DMSO.Total proteins were extracted from the cells treated with 0.2% DMSO ( control) or 20 μmol /L meisoindigo ( Test) for 2 hours.Then,the TMT-labeling HPLC - MS /MS was used to identify and quantify the peptides and their abundance,all the tests were repeated for 3 times.The Mascot software was used to identify the proteins; the GO annotations,enrichment and cluster analysis were used to analyze the differentially expressed proteins.Results: Meisoindigo-induced K562 cell apoptosis in a dose-dependent manner ( r=0.98) ,5 544 proteins were identified,4792 of which were quantified.The protein with expression difference >1.5 - folds in Test group accoanted for 8,out of which the expression of 4 proteins were up - regulated and 4 were down - regulated.The differentially expressed proteins mainly associated with reactive oxygen species ( ROS) .Conclusion: Several proteins including DDIT4 were found to have dramatic changes in the early stage of K562 cells treated with meisoindigo by using quantitative proteomics technology.The ROS metabolic process may play important roles in meisoindigo - inducing apoptosis of K562 cells.

关 键 词：慢性粒细胞白血病 K562 甲异靛 定量蛋白质组学 

分 类 号：R733.72[医药卫生—肿瘤]