细粒棘球蚴原头节分泌的细胞外囊泡中人源蛋白质组学分析

Proteomic analysis of human proteins in extracellular vesicles secreted by protoscoleces of Echinococcus granulosus

目的蛋白质组学分析鉴定来源于细粒棘球蚴原头节分泌的细胞外囊泡(EVs)中人源蛋白质的组分。方法收集细粒棘球蚴病患者肝脏中的完整包囊,无菌条件下分离原头节,用含30%去外泌体的胎牛血清完全培养基体外培养原头节,每3天换1次培养基并收集培养上清,用EVs分离试剂盒分离培养上清中的EVs。透射电镜观察EVs的形态;纳米粒子跟踪分析EVs的粒径;采用液相色谱-质谱法对EVs进行蛋白质组学测序,二级质谱数据使用Maxquant(v1.5.2.8)进行蛋白检索与注释。将人源蛋白通过在线工具David进行组织、疾病及通路的富集比对与注释。结果体外培养原头节分泌的EVs为杯状双层膜性囊泡,直径小于200 nm。蛋白质组学测序共测出20317条肽段,鉴定获得1461个蛋白,其中328个为人源蛋白。原头节分泌的EVs中高度富集的外泌体标志蛋白有细丝蛋白、14-3-3蛋白、热休克蛋白90、热休克蛋白70和丙酮酸激酶。EVs的人源蛋白中,组织富集程度居前3位的为血浆、Cajal-Retzius细胞、肝脏。疾病富集中涉及感染、免疫和癌症等相关疾病。Bio Carta通路富集结果显示,富集程度居前3位的分别为替代补体通路、凝集素补体通路和补体通路;KEGG通路富集程度居前3位的为黏着斑黏附信号通路、细胞外基质受体相互作用通路和蛋白酶体相互作用通路。结论体外培养原头节分泌的EVs中的人源蛋白与人体血浆和肝脏等组织相关,提示原头节通过分泌EVs与人体进行物质交换。

Objective To identify the protein of human source in extracellular vesicles(EVs)secreted by protoscoleces of Echinococcus granulosus by proteomic analysis.Methods Liver intact cysts were collected from cystic echinococcosis patients,and the protoscoleces were separated under aseptic conditions and then cultured in 30%exosome-free fetal bovine serum in vitro,with medium replacement every three days.The supernatant was collected,from which EVs were isolated by a simple and rapid extracellular vesicle separation kit.A transmission electron microscope was employed to observe and identify the morphology of EVs,and the concentration and size of EVs were evaluated by nanoparticle tracking analysis.The EVs underwent Liquid chromatography-mass spectrometry analysis for identification and characterization of their protein content.The secondary mass spectrometry data were used to retrieve and annotated with Maxquant(v1.5.2.8).The enrichment analysis of tissues,disease and pathway for the human source proteins in EVs were were carried out using online tool David.Results The EVs secreted by protoscoleces in vitro were cup-shaped bilayer membranous vesicles with a diameter less than 200 nm.The proteomic analysis found a total of 20317 peptides,and identified 1461 proteins,including 328 human source proteins.Exosome marker proteins,including filament protein,14-3-3 protein,heat shock protein 90,heat shock protein 70 and pyruvate kinase,are highly enriched in EVs secreted by protoscolex.The top 3 tissues in enrichment extent were plasma,Cajal-Retzius cells and liver.The disease enrichment found involving infection,immunity and cancer.The Bio Carta pathways analysis indicated top 3 enriched pathways,which were the alternative complement pathway,lectin complement pathway and complement pathway;the top 3 of KEGG pathways enrichment were adhesion plaque,extracellular matrix receptor-related action,and proteasome.Conclusion The proteins of human source in EVs secreted by protoscoleces cultured in vitro are related to human plasma and liver,indicating that the protoscoleces exert substance exchange with human via seretion of EVs.