脊髓损伤患者血浆外泌体蛋白分子标志物的筛选研究

Study of molecular markers of plasma exosomal proteins in patients with spinal cord injury

目的应用蛋白质非标记定量技术和生物信息学分析筛查脊髓损伤(spinal cord injury,SCI)患者血浆外泌体蛋白分子标志物。方法收集2021年1月至2022年6月南京医科大学第一附属医院SCI患者及健康对照人群血浆标本各50份。使用超速离心法分离血浆外泌体,通过透射电子显微镜、纳米粒子跟踪分析和Western blot对外泌体进行鉴定。利用Label-Free定量蛋白质组学分析血浆外泌体差异表达蛋白(differentially expressed proteins,DEPs),并基于基因本体(Gene Ontology,GO)和基因功能和基因组信息(kyoto encyclopedia of genes and genomes,KEGG)数据库对DEPs进行表征、注释和富集分析。利用血浆外泌体标本对筛选出的DEPs进行Western blot和酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)验证。结果根据美国脊柱损伤协会脊髓损伤分级:A级14例、B级19例、C级12例、D级5例。SCI患者的血浆外泌体与对照组外泌体均呈现典型的杯状形态,直径30~200 nm。经蛋白质非标记定量技术共鉴定出493种外泌体蛋白,筛选出126种蛋白质差异表达,其中38种上调、88种下调。GO注释显示了DEPs主要参与蛋白质激活级联、补体激活和免疫反应等功能。KEGG富集分析显示了DEPs参与补体和凝血级联反应、蛋白酶体和神经退行性疾病通路等生物学途径。根据蛋白质组学和生物信息学分析初步筛选出2个候选蛋白:APOB和S100A9。Western blot结果显示30份SCI患者血浆外泌体中S100A9蛋白相对表达量(1.62±0.19)较30份对照组(0.86±0.24)升高,差异有统计学意义(t=8.55,P<0.001),而APOB蛋白相对表达量(1.06±0.13和1.02±0.23)比较差异无统计学意义(t=0.46,P=0.653)。ELISA试验结果显示S100A9在不同程度SCI患者血浆外泌体中表达[A级(197.7±11.7)pg/ml、B级(151.7±15.2)pg/ml、C级(136.3±14.7)pg/ml]差异有统计学意义(F=69.94,P<0.001),SCI严重程度越高,S100A9在血浆外泌体中的表达越高(A级与B级,q=13.11,P<0.001;A级与C级,q=15.66,P<0.001;B级与C级,q=4.19,P=0.005)。结论S100A9是评估SCI严重程度的潜在有效血浆外泌体分子标志物。

Objective To screen plasma exosomal protein molecular markers in patients with spinal cord injury(SCI)by applying Label-Free quantification and bioinformatics analysis.MethodsFifty plasma specimens from the First Affiliated Hospital of Nanjing Medical University(from January 2021 to June 2022)were collected from SCI patients and healthy people,respectively.Plasma exosomes were isolated using ultracentrifugation and identified by transmission electron microscopy,nanoparticle tracking analysis and western blot.Plasma exosomal differentially expressed proteins(DEPs)were analyzed using Label-Free quantitative proteomics,and DEPs were characterized,annotated,and enriched based on Gene Ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG)databases.The screened DEPs were validated by western blot and enzyme linked immunosorbent assay(ELISA)using plasma exosomal specimens.ResultsAccording to the spinal cord injury classification of the American Spinal Injury Association,14 cases were grade A,19 cases were grade B,12 cases were grade C,and 5 cases were grade D.Plasma exosomes of SCI patients and control groups showed typical cup-like morphology,with diameters mainly ranging from 30-200 nm.A total of 493 exosomal proteins were identified by Label-Free quantification,and 126 proteins were screened for differential expression,of which 38 were up-regulated and 88 were down-regulated.GO annotation revealed that DEPs were mainly involved in functions such as protein activation cascade,complement activation and immune response.KEGG pathway analysis revealed that DEPs were involved in biological pathways such as complement and coagulation cascade reactions,proteasome and neurodegenerative disease pathways.Two candidate proteins,APOB and S100A9,were initially screened based on quantitative results from proteomics and bioinformatics analyses.Western blot results showed that the relative expression of S100A9 protein in plasma exosomes of 30 SCI patients(1.62±0.19)was elevated compared with that of 30 control groups(0.86±0.24),and the difference was statistically significant(t=8.55,P<0.001),while the relative expression of APOB protein(1.06±0.13 and 1.02±0.23)were not statistically significant(t=0.46,P=0.653).The results of ELISA analysis showed that the expression of S100A9 in plasma exosomes of patients with different degrees of SCI(grade A 197.7±11.7 pg/ml,grade B 151.7±15.2 pg/ml,grade C 136.3±14.7 pg/ml)had statistical significance(F=69.94,P<0.001),the higher the severity of SCI,the higher the expression of S100A9 in plasma exosomes(A vs.B,q=13.11,P<0.001;A vs.C,q=15.66,P<0.001;B vs.C,q=4.19,P=0.005).ConclusionS100A9 is a potentially valid plasma exosomal molecular marker for assessing the severity of SCI.