基于蛋白质组学对脊柱结核患者外周血浆差异蛋白筛选及功能分析

Screening and functional analysis of differential proteins in peripheral plasma of spinal tuberculosis patients based on proteomics

中文关键词:  脊柱结核  蛋白质组学  差异蛋白  信号通路

英文关键词:Spinal tuberculosis  Proteomics  Differential protein  Signaling pathway

中文摘要:

  【摘要】 目的：利用蛋白质组学技术筛选脊柱结核（spinal tuberculosis，STB）患者外周血浆中表达差异的蛋白质，探讨STB致病机制及代谢途径。方法：收集30例STB患者和30例健康志愿者的外周血，取其中3例STB患者（观察组）和3例健康志愿者（对照组）的外周血浆通过蛋白质组学同位素标记相对及绝对定量技术和液相色谱-质谱分析技术鉴定差异蛋白，筛选出表达差异显著（差异倍数>1.2，P<0.05）的蛋白质，并进行基因本体（gene ontology，GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析；然后将30例STB患者和30例健康志愿者的血浆进行实时荧光定量逆转录聚合酶链反应（real-time quantitative polymerase chain reaction，RT-PCR）和酶联免疫吸附测定法（enzyme-linked immunosorbent assay，ELISA）验证其差异基因及蛋白。结果：STB患者外周血浆中共筛选出16个与对照组存在显著性差异的蛋白；其中11个蛋白[脂多糖蛋白（lipopolysaccharide-binding protein，LBP）、S100-A8蛋白（protein S100-A8）、S100-A9蛋白（protein S100-A9）、淀粉样蛋白A-2（serum amyloid A-2 protein，SAA2）、富含亮氨酸α-2糖蛋白（leucine-rich alpha-2-glycoprotein，LRG2）、蛋白聚糖4（proteoglycan 4，PRG4）、补体因子Ⅰ（complement factor I，CFI）、α-1-抗胰蛋白酶（alpha-1-antitrypsin，SERPINA1）、组织蛋白酶D（cathepsin D，CTSD）、血浆铜蓝蛋白（ceruloplasmin，CP）和λ免疫球蛋白（lambda-immunoglobulin 4-60，IGLV4-60）]表达上调，5个蛋白[黏着斑蛋白（vinculin，VCL）、3-64D免疫球蛋白（immunoglobulin heavy variable 3-64D，IGHV3-64D）、2D-29免疫球蛋白（immunoglobulin kappa variable 2D-29，IGKV2D-29）、非功能性免疫球蛋白（non-functional immunoglobulin 6D-41，IGKV6D-41）和1-45免疫球蛋白（immunoglobulin heavy variable 1-45，IGHV1-45）]表达下调。LBP、S100-A8蛋白和S100-A9蛋白在STB患者外周血浆的基因和蛋白表达与对照组比较均显著性升高（P＜0.05），主要参与白介素-17信号通路和自噬信号通路，且在以上信号通路中与炎症、自噬、趋化因子等生物学功能相关。SAA2、LRG2、PRG4、CFI、SERPINA1、CTSD、CP和VCL的基因和蛋白表达与对照组无统计学差异；IGLV4-60、IGHV3-64D、IGKV2D-29、IGKV6D-41和IGHV1-45的基因表达与对照无显著性差异（P＞0.05），蛋白表达未验证。结论：蛋白质组学可用于STB患者外周血差异蛋白的筛选及STB发病机制的研究，LBP、S100-A8和S100-A9蛋白可能是STB发病相关的重要表达蛋白，主要参与IL-17信号通路和自噬相关信号通路且集中于炎症、自噬、趋化因子等生物学功能。

英文摘要:

  【Abstract】 Objectives: To screen the differentially expressed proteins in peripheral plasma of patients with spinal tuberculosis with proteomics technology to provide reference for the study of the pathogenic mechanism and metabolic pathway of spinal tuberculosis. Methods: Peripheral blood was collected from 30 patients with spinal tuberculosis and 30 healthy volunteers, and the peripheral plasma from three patients with spinal tuberculosis(observation group) and three healthy volunteers(control group) was identified by isobaric tags for relative and absolute quantification(iTRAQ) based proteomics technique and liquid chromatography-mass spectrometry analysis for differential proteins to screen the proteins with significant expression differences(FC>1.2, P<0.05), and enrichment analysis was performed through Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG). Then, plasma from spinal tuberculosis patients and healthy volunteers was tested by real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA) to verify the differential genes and proteins. Results: A total of 16 proteins were screened in the peripheral plasma of patients with spinal tuberculosis that were significantly different from those of the control group. Among them, 11 were up-regulated[lipopolysaccharide-binding protein(LBP), S100-A8, S100-A9, serum amyloid A-2 protein(SAA2), leucine-rich alpha-2 glycoprotein(LRG2), proteoglycan 4 (PRG4), complement factor I(CFI), alpha-1-antitrypsin(SERPINA1), cathepsin D(CTSD), ceruloplasmin(CP), and lambda-immunoglobulin variable 4-60(IGLV4-60)] and 5 proteins were down-regulated[vinculin(VCL), immunoglobulin heavy variable 3-64D(IGHV3-64D), immunoglobulin kappa variable 2D-29(IGKV2D-29), non-functional immunoglobulin kappa variable 6D-41(IGKV6D-41), and immunoglobulin heavy variable 1-45(IGHV1-45)]. The gene and protein expressions of LBP, S100-A8, and S100-A9 proteins were significantly elevated in the peripheral plasma of patients with spinal tuberculosis compared with those of control group(P<0.05). They mainly involved in the interleukin-17 signaling pathway and autophagy signaling pathway, where they were associated with inflammation, autophagy, chemokines and other biological functions. The gene and protein expressions of SAA2, LRG2, PRG4, CF1, SERPINA1, CTSD, CP, and VCL were not statistically different from those of the control group; the gene expression of IGLV4-60, IGHV3-64D, IGKV2D-29, IGKV6D-41, and IGHV1-45 were not significantly different from those of the control group(P>0.05), and the protein expression was not validated. Conclusions: Proteomics can be used to screen differential proteins in peripheral blood of patients with spinal tuberculosis and study the pathogenesis of spinal tuberculosis. LBP, S100-A8 and S100-A9 proteins may be important expression proteins related to the pathogenesis of spinal tuberculosis, which proteins mainly participate in IL-17 signaling pathway and autophagy-related signal pathway, and focus on biological functions such as inflammation, autophagy and chemokines.