非标记定量蛋白质组学技术探讨不同透析龄患者腹膜透析流出液外泌体差异蛋白的研究
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E-mail:lizhang6@126.com

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R586

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江苏省医学创新团队(CXTDA2017011)


The study of label ⁃free quantitative proteomics technology on the differential proteins of exosomes in peritoneal dialysis effluent from patients with different dialysis ages
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    目的:研究不同透析龄患者腹膜透析流出液(peritoneal dialysis effluent,PDE)外泌体中差异蛋白质组。方法:选取 10例稳定腹膜透析(peritoneal dialysis,PD)患者,根据透析龄分为初始腹透组(n=5)和维持腹透组(n=5)。收集患者留腹过夜的PDE,通过透射电镜(transmission electron microscopy,TEM)、纳米颗粒跟踪分析(nanoparticle tracking analysis,NTA)、免疫印迹法鉴定经超速离心提取的外泌体,并用非标记定量蛋白质组学技术鉴定和筛选,对具有显著差异的蛋白开展功能富集和信号通路生物信息学分析。结果:PDE中提取到的外泌体TEM下呈茶托状,直径分布在30~150 nm,表达外泌体标志蛋白CD63 和TSG101。通过数据库比对两组共筛选出499种差异蛋白,与初始腹透组相比,维持腹透组经差异显著性筛选上调蛋白17 种,下调蛋白15种。基因本体论(gene ontology,GO)分析显示,差异表达蛋白主要分布在氯离子通道复合物、细胞表面以及核基质,以结合与催化活性为主,参与结合的正向调控、氧化应激反应及过氧化物酶反应等生物过程。京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路主要富集在免疫相关的信号通路,包括IL⁃17和Th17细胞分化信号通路,在腹膜损伤中起着关键作用。结论:采用非标记定量蛋白质组学技术筛选的差异蛋白可能作为PD患者腹膜损伤的候选标志物,也为揭示腹膜纤维化分子生物学机制提供线索。

    Abstract:

    Objective:To study the differential proteomics in exosomes of peritoneal dialysis effluent(PDE)from patients with different dialysis ages. Methods:Ten stable peritoneal dialysis(PD)patients were selected and divided into newly enrolled patients group(NEPs group,n=5)and maintenance peritoneal dialysis patients group(MPDs group,n=5)according to the dialysis age. PDE was collected from patients left overnight. Exosomes were extracted by ultracentrifugation and identified by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA),and Western blotting. Label ⁃free quantitative proteomics technology was used to identify and screen the exosomes. Functional enrichment and signal pathway bioinformatics analysis were performed for significantly differential proteins in the exosomes. Results:The exosomes in PDE showed saucer ⁃like vesicles under TEM,and the diameters were between 30 and 150 nm measured by NTA. The specific markers of exosomes,CD63 and TSG101 were detected by Western blotting. A total of 499 proteins were detected by proteomics analysis. After significant difference screening,17 were up ⁃ regulated and 15 were down ⁃ regulated. Gene ontology(GO)analysis showed that these differentially expressed proteins were mainly distributed in chloride channel complexes,cell surface and nuclear matrix,playing a role mainly in binding and catalytic activities,and were involved in biological processes such as positive regulation of binding,oxidative stress response and peroxidase response. Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were mainly enriched in immunE-related signaling pathways,including IL⁃17signaling pathway and Th17 cell differentiation signaling pathway,which play a key role in peritoneal injury. Conclusion:The differentially expressed proteins screened by label ⁃ free quantitative proteomics technology could be used as candidate markers of peritoneal injury in PD patients and provids molecular clues for revealing the peritoneal fibrosis mechanism of pathogenesis.

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武云慧,黄抱娣,茅春霞,李归雁,邢昌赢,张莉.非标记定量蛋白质组学技术探讨不同透析龄患者腹膜透析流出液外泌体差异蛋白的研究[J].南京医科大学学报(自然科学版),2022,42(8):1133-1141

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  • 在线发布日期: 2022-08-07
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