活动性肺结核患者外周血CD4~+T淋巴细胞的label-free蛋白组学初步分析
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摘要
目的应用非标记label-free蛋白质组学方法筛选出活动性肺结核患者外周血CD4~+T淋巴细胞差异表达蛋白质,为阐明结核病的发病机制和早期诊断提供理论依据。方法收集分离9例活动性肺结核患者和9例健康人外周血CD4~+T淋巴细胞,应用label-free非标记蛋白组学方法检测外周血CD4~+T淋巴细胞蛋白质谱,筛选出差异表达蛋白质。并用基因本体论(GO)及京都基因与基因组百科全书(KEGG)生物信息学分析软件对差异表达蛋白质进行分析。结果活动性肺结核与健康人外周血CD4~+T淋巴细胞中的蛋白质组分布的基本框架很相似,但可发现二者之间有38个明显差异表达的蛋白点,其中有26种蛋白质在肺结核组中表达上调(差异倍数>1.5,P<0.05),12种蛋白质在肺结核组中表达下调(差异倍数<0.67,P<0.05)。GO分析结果表明,大部分的差异蛋白质主要定位于胞内区域,结核感染相关差异蛋白质功能体现在结合、调控以及代谢过程中。KEGG分析表明,磷酸戊糖途径、cGMP/PKG信号通路、磷脂酰肌醇信号系统等与结核分枝杆菌感染密切相关。结论对活动性肺结核患者外周血CD4~+T淋巴的比较蛋白质组学研究一共鉴定出38种差异蛋白质,他们可能在肺结核发病过程中起着重要作用,也可能可做为诊断结核病的潜在生物学标志物。
Objective active pulmonary tuberculosis(APTB)by label-free quantitative proteomics,and to lay foundation for tuberculosis pathogenesis,diagnosis and therapy.Methods CD4~+T cells were collected and purified from 9 patients with tuberculosis and9 healthy controls.A label-free quantitative proteomic strategy was used to identify the differently expressed proteins between the two groups.Subsequent bioinformatics analyses of these differential expressed proteins were done by GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes).Results The basic frames of proteomic distribution of CD4~+T cells were very similar,but 38 differential protein spots were found in these two types.Among these 38 protein spots,26 spots were highly expressed and 12 poorly expressed in CD4~+T cells of APTB(>1.5 or<0.67 fold,P<0.05).Gene Ontology(GO)analysis revealed most differently expressed proteins had a intracellular distribution.MTB infection was mainly related to differences in binding,regulating and metabolic processes.KEGG enrichment analysis indicated that pentose phosphate pathway,cGMP-PKG signaling pathway,phosphalidylinositol signaling system etc.were significantly associated with the Mtb infection.ConclusionsA total of 38 proteins are identified by comparative proteomics analysis of differential proteins expression in CD4~+T cells from patients with pulmonary tuberculosis,which may have an important role in the pathogenesis of pulmonary tuberculosis and may be a potential biomarker for tuberculosis diagnosis.
Objective active pulmonary tuberculosis(APTB)by label-free quantitative proteomics,and to lay foundation for tuberculosis pathogenesis,diagnosis and therapy.Methods CD4~+T cells were collected and purified from 9 patients with tuberculosis and9 healthy controls.A label-free quantitative proteomic strategy was used to identify the differently expressed proteins between the two groups.Subsequent bioinformatics analyses of these differential expressed proteins were done by GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes).Results The basic frames of proteomic distribution of CD4~+T cells were very similar,but 38 differential protein spots were found in these two types.Among these 38 protein spots,26 spots were highly expressed and 12 poorly expressed in CD4~+T cells of APTB(>1.5 or<0.67 fold,P<0.05).Gene Ontology(GO)analysis revealed most differently expressed proteins had a intracellular distribution.MTB infection was mainly related to differences in binding,regulating and metabolic processes.KEGG enrichment analysis indicated that pentose phosphate pathway,cGMP-PKG signaling pathway,phosphalidylinositol signaling system etc.were significantly associated with the Mtb infection.ConclusionsA total of 38 proteins are identified by comparative proteomics analysis of differential proteins expression in CD4~+T cells from patients with pulmonary tuberculosis,which may have an important role in the pathogenesis of pulmonary tuberculosis and may be a potential biomarker for tuberculosis diagnosis.
引文
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[16]易平,田芳,郭旭丽,等.外周血T细胞抗原刺激试验(T-SPOT)在结核病检测中的应用[J].实用预防医学,2017,24(3):260-264.
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[20]KUMAR N P,MOIDEEN K,GEORGE P J,et al.Coincident diabetes mellitus modulates Th1-,Th2-,and Th17-cell responses in latent tuberculosis in an IL-10-and TGF-β-dependent manner[J].Eur J Immunol,2016,46(2):390-399.
[2]叶晓雪,许崇.多层螺旋CT在肺结核诊断及分型中的应用价值[J].医学影像学杂志,2014,24(2):321-322.
[3]姚雪云.使用低剂量螺旋CT技术和X线胸片技术诊断肺结核的效果对比[J].影像研究与医学应用,2018,2(2):110-111.
[4]LA MANNA M P,ORLANDO V,LI DONNI P,et al.Identification of plasma biomarkers for discrimination between tuberculosis infection/disease and pulmonary non tuberculosis disease[J].PLoSONE,2018,13(3):e0192664.
[5]ZAMBUZI F A,CARDOSO-SILVA P M,ESPINDOLA M S,et al.Identification of promising plasma immune biomarkers to differentiate active pulmonary tuberculosis[J].Cytokine,2016,88:99-107.
[6]JACOBS R,MALHERBE S,LOXTON A G,et al.Identification of novel host biomarkers in plasma as candidates for the immunodiagnosis of tuberculosis disease and monitoring of tuberculosis treatment response[J].Oncotarget,2016,7(36):57581-57592.
[7]DEBORAH A L,GWENDOLYN M S,BYUNG P,et al.Comprehensive definition of human immunodominant CD8 antigens in tuberculosis[J].NPJ Vaccines.2017,2(1):8.
[8]JASENOSKY L D,SCRIBA T J,HANEKOM W A,et al.T cells and adaptive immunity to Mycobacterium tuberculosis in humans[J].Immunol Rev,2015,264(1):74-87.
[9]SANDBERG A,LINDELL G,K?LLSTR?M B N,et al.Tumor proteomics by multivariate analysis on individual pathway data for characterization of vulvar cancer phenotypes[J].Mol Cell Proteomics,2012,11(7):M112.016998.
[10]李靖,何艳,郑煜煌,等.肺结核患者Th17和调节性T细胞在抗结核治疗过程中的变化[J].中华微生物学和免疫学杂志,201232(9):813-815.
[11]梁友宝,梁华,王兆华,等.肺结核患者外周血不同T淋巴细胞亚群中白细胞介素-9产生细胞比例的研究[J].国际免疫学杂志2017,40(2):123-126.
[12]储开枫.结核感染T细胞斑点试验对肺结核的诊断意义分析[J]饮食保健,2018,5(1):250-251.
[13]林永通,麦世康.GeneXpert Mtb/RIF检测技术在结核病诊断中的应用评价[J].中国热带医学,2018,18(1):93-95.
[14]LAURENTI P,RAPONI M,DE WAURE C,et al.Performance of interferon-γrelease assays in the diagnosis of confirmed active tuberculosis in immunocompetent children:a new systematic review and meta-analysis[J].BMC Infect Dis,2016,16:131.
[15]陈芳,项忠元.T淋巴细胞干扰素释放试验在检测结核杆菌感染中的应用[J].实用预防医学,2017,24(3):272-274.
[16]易平,田芳,郭旭丽,等.外周血T细胞抗原刺激试验(T-SPOT)在结核病检测中的应用[J].实用预防医学,2017,24(3):260-264.
[17]WOODWORTH J S,AAGAARD C S,HANSEN P R,et al.Protective CD4 T cells targeting cryptic epitopes of Mycobacterium tuberculosis resist infection-driven terminal differentiation[J].JImmunol,2014,192(7):3247-3258.
[18]MAYER-BARBER K D,BARBER D L.Innate and Adaptive Cellular Immune Responses toMycobacterium tuberculosisInfection[J].Cold Spring Harbor Perspectives in Medicine,2015:a018424.
[19]RIOU C,STRICKLAND N,SOARES A P,et al.HIV Skews the Lineage-Defining Transcriptional Profile of Mycobacterium tuberculosis-Specific CD4+T Cells[J].J Immunol,2016,196(7):3006-3018.
[20]KUMAR N P,MOIDEEN K,GEORGE P J,et al.Coincident diabetes mellitus modulates Th1-,Th2-,and Th17-cell responses in latent tuberculosis in an IL-10-and TGF-β-dependent manner[J].Eur J Immunol,2016,46(2):390-399.