去白细胞单采血小板保存期外泌体形态及蛋白质组学分析  

Morphology and proteomic analysis of leukocyte-free apheresis-derived exosome in storage

作　　者：谢月娜 赵倩 李静 张佳慧 李凤园 种靖慧 XIE Yuena;ZHAO Qian;LI Jing;ZHANG Jiahui;LI Fengyuan;CHONG Jinghui(Tianjin Blood Center,Tianjin 300110,China)

机构地区：[1]天津市血液中心,天津300110

出　　处：《中国输血杂志》2024年第10期1101-1109,共9页Chinese Journal of Blood Transfusion

摘　　要：目的探讨去白细胞单采血小板(leukocyte-free apheresis platelet,LFA-Plt)保存期内所释放的外泌体(exosome,EXO)形态及蛋白组学变化,评价血小板保存质量,预测不同保存期血小板EXO功能。方法超速离心法提取EXO,并对其进行电镜、粒径、WB鉴定;4D Label-free定量蛋白质组学技术对EXO进行蛋白定量分析,并对所有鉴定到的蛋白质进行生物信息学分析;对保存d3、d5两组LFA-Plt EXO进行蛋白差异分析并对差异蛋白进行GO功能和KEGG通路显著性富集分析。结果成功获取杯托状、CD9/TSG101富集、Calnexin(-)的EXO;保存d3、d5的LFA-Plt EXO粒径分别为(82.2±19.6)nm、(83.4±19.4)nm;蛋白浓度分别为(0.55±0.13)μg/μL、(0.51±0.08)μg/μL,差异无统计学意义(P>0.05);两组样品中共鉴定到1504个蛋白质,GO功能富集分析显示,LFA-Plt所分泌的EXO蛋白主要集中在细胞膜、胞外区以及蛋白酶体核心复合体,与蛋白、ATP、钙离子及GTP结合能力相关,主要参与氧化还原、蛋白质水解、信号转导等过程;KEGG功能注释显示,EXO蛋白主要参与物质运输、分解代谢及遗传信息的处理,与人类肿瘤及病毒细菌感染密切相关,影响人类免疫系统的代谢;与d3相比,d5 EXO有16个蛋白显著上调;GO富集分析显示16种上调蛋白主要与腺苷同型半胱氨酸活性、6-磷酸果糖激酶活性相关,主要参与有机氮化合物代谢过程;KEGG富集通路分析显示保存末期LFA-Plt EXO上调的蛋白最主要功能是参与孕酮介导的卵母细胞成熟的信号通路。结论本中心LFA-Plt制备、保存条件下血小板质量能够保持相对稳定;不同保存期EXO蛋白质功能有一定差别,可能影响血小板输注效果。Objective To investigate the morphological and proteomic differences in exosomes(EXOs)during the storage of leukocyte-free apheresis platelets(LFA-Plt),evaluate the quality of platelets in storage and predict the function of EXOs at different storage periods.Methods EXOs were isolated by ultracentrifugation,then the morphological observation was performed by electron microscope.Particle size analysis and WB protein index detection were performed.4D Label-free quantitative proteomics technology was used to perform quantitative and bioinformatics analysis on identified proteins.Protein differential analysis on the LFA-Plt EXO between group day 3 and day 5 was performed,and GO function and KEGG pathway enrichment analysis on differential proteins was conducted.Results Cup shaped,CD9/TSG101 enriched and Calnexin(-)EXO was successfully obtained.The particle size(nm)of LFA-Plt EXO for day 3 and day 5 were(82.2±19.6)and(83.4±19.4)respectively,and the protein concentration(μg/uL)were(0.55±0.13)and(0.51±0.08)respectively,with no statistically significant difference between two groups(P>0.05).1504 proteins were identified in all samples.GO functional enrichment analysis showed that the LFA-Plt EXO proteins were mainly concentrated in the cell membrane,extracellular domain and proteasome core complex,and were related to the binding ability of proteins,ATP,calcium ions and GTP,and mainly participated in processes such as redox,protein hydrolysis and signal transduction.KEGG functional annotation showed that the EXO proteins mainly participated in material transportation and catabolism,genetic information processing,and were closely related to human tumors and viral bacterial infections,affecting the metabolism of human immune system.Compared with day 3,day 5 EXO showed significant up-regulation in 16 proteins.The GO enrichment analysis showed that 16 upregulated proteins were mainly associated with adenosine homocysteine activity and 6-phosphofructose kinase activity,and were mainly involved in the metabolism of organic

关 键 词：去白细胞单采血小板 保存期 外泌体 蛋白质组学 

分 类 号：R457.12[医药卫生—治疗学] R331.14[医药卫生—临床医学] Q816[生物学—生物工程]