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

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。

Label-free quantitative proteomics analysis of proteins in peripheral blood CD4+ T cells from patients with active pulmonary tuberculosis

Objective To screen the differential expressed proteins of peripheral blood CD4⁺ T cells in patients with 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 and 9 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 1poorly 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. Conclusions A 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．