Page 91 - 南京医科大学学报自然科学版

第42卷第8期 南京医科大学学报（自然科学版）
2022年8月 Journal of Nanjing Medical University（Natural Sciences） ·1133 ·

·临床研究·

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

武云慧 ，黄抱娣 ，茅春霞 ，李归雁 ，邢昌赢 ，张 莉 1*
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南京医科大学第一附属医院肾内科，营养科，江苏 南京 210029
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［摘 要］ 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。
［关键词］ 腹膜透析；流出液；外泌体；蛋白质组学；非标记技术
［中图分类号］ R586 ［文献标志码］ A ［文章编号］ 1007⁃4368（2022）08⁃1133⁃09
doi：10.7655/NYDXBNS20220813

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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WU Yunhui ，HUANG Baodi ，MAO Chunxia ，LI Guiyan ，XING Changying ，ZHANG Li 1*
Department of Nephrology，Department of Nutrition，the First Affiliated Hospital of Nanjing Medical University，
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Nanjing 210029，China
［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⁃17

［基金项目］ 江苏省医学创新团队（CXTDA2017011）
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通信作者（Corresponding author），E⁃mail：lizhang6@126.com