细胞因子IL-17作为牛结核病诊断标志物的研究及实时荧光定量PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2018.06.003

摘要/Abstract

摘要：

试验旨在评价细胞因子IL-6和IL-17 mRNA转录水平与牛分枝杆菌感染之间的关系，及其在牛结核病诊断中的应用潜力。通过皮内变态反应试验和IFN-γ释放试验临床筛选结核病阳性牛和结核病阴性牛，采集试验动物抗凝全血，分离、收集外周血淋巴细胞，分别用牛结核菌素（PPD-B）、禽结核菌素（PPD-A）、重组蛋白CFP-10-ESAT-6（CE）、pET-32a载体标签蛋白（PET）或PBS 37℃培养6 h，用实时荧光定量PCR检测细胞因子IL-6、IL-17和IFN-γ的mRNA相对转录水平。结果显示，PET和空白对照PBS类似，不能刺激细胞因子mRNA转录水平的提高，表明CE中包含的PET对试验的影响可忽略不计；牛外周血淋巴细胞经PPD-B、PPD-A或CE刺激后，结核病阳性牛样品中IL-17和IFN-γ的mRNA转录水平均显著高于结核病阴性牛（P<0.05），其中PPD-B刺激效果强于CE和PPD-A，而CE刺激的特异性更好；选取CE作为最佳刺激源，结果显示，IL-17和IFN-γ的mRNA转录水平之间相关性良好（spearman r=0.79），并初步建立了基于IL-17和IFN-γ转录水平的实时荧光定量PCR检测方法；以此方法对14头结核病阳性牛进行临床检验，IL-17实时荧光定量PCR法的阳性样本检出率为85.7%，高于IFN-γ（71.4%）。本研究结果初步表明，牛分枝杆菌特异性抗原（PPD-B、CE）诱导的IL-17 mRNA转录水平与牛结核病相关，以CE为刺激源建立的IL-17实时荧光定量PCR检测方法具有用于牛结核病诊断的潜力。

关键词: 牛结核病; 诊断; IL-17; IFN-γ ; 实时荧光定量PCR

Abstract:

This study was aimed to explore the correlation between the transcription levels of cytokines (IL-6 and IL-17) and the infection of Mycobacterium bovis,and evaluate potential of these cytokines for bovine tuberculosis (bTB) diagnosis.Clinically TB-infected cattle and healthy cattle were screened by the skin tests and IFN-γ release assays.Heparinized blood from each animal was collected and used for isolation of the peripheral blood lymphocytes (PBMC).PBMCs were respectively incubated with bovine tuberculin (PPD-B),avian tuberculin (PPD-A),recombinant protein CFP-10-ESAT-6 (CE),PET or PBS at 37℃ for 6 h,and then the transcription levels of IL-6,IL-17 and IFN-γ mRNA were detected by Real-time PCR.As with PBS,PET could not stimulate the increase of cytokine mRNA level,which indicated the influence of PET contained in CE was negligible.PPD-B,PPD-A or CE significantly increased mRNA levels of IL-17 and IFN-γ in PMBCs from TB-infected cattle (P<0.05), of which PPD-B showed the highest stimulus intensity,while CE exhibited the best stimulus specificity.Under the condition that CE was selected as optimal stimulus,the correlation between IL-17 and respective IFN-γ mRNA levels were observed (spearman r=0.79),and then Real-time PCR assays of IL-17 and IFN-γ were preliminary developed.The detection rate of IL-17-based method on 14 clinical bTB-positive samples was 85.7%,which was higher than 71.4% of IFN-γ.Taken together,these findings supported the IL-17 mRNA level in PBMC was related with bTB infection,and demonstrated that measurement of IL-17 mRNA by Real-time PCR was potential for bTB diagnosis.

Key words: bovine tuberculosis; diagnosis; IL-17; IFN-γ; Real-time PCR

中图分类号:

S855.2

高新桃, 贾红, 侯绍华, 郭晓宇, 袁维峰, 姜一曈, 朱鸿飞, 鑫婷. 细胞因子IL-17作为牛结核病诊断标志物的研究及实时荧光定量PCR检测方法的建立[J]. 《中国畜牧兽医》, 2018, 45(6): 1447-1453.

GAO Xintao, JIA Hong, HOU Shaohua, GUO Xiaoyu, YUAN Weifeng, JIANG Yitong, ZHU Hongfei, XIN Ting. Research on the Potential of IL-17 as a Biomarker of Bovine Tuberculosis and Establishment of Its Real-time PCR Assay[J]. , 2018, 45(6): 1447-1453.

参考文献

[1] SMITH N H,KREMER K,INWALD J,et al.Ecotypes of the Mycobacterium tuberculosis complex[J].Journal of Theoretical Biology,2006,239(2):220-225.
[2] DOYLE L P,COURCIER E A,GORDON A W,et al.Bovine tuberculosis in Northern Ireland:Quantification of the population disease-level effect from cattle leaving herds detected as a source of infection[J].Epidemiology and Infection,2017,145(16):3505-3515.
[3] SCHILLER I,OESCH B,VORDERMEIER H M,et al.Bovine tuberculosis:A review of current and emerging diagnostic techniques in view of their relevance for disease control and eradication[J].Transboundary and Emerging Diseases,2010,57(4):205-220.
[4] JIANG T T,SHI L Y,WEI L L,et al.Serum amyloid A,protein Z,and C4b-binding protein β chain as new potential biomarkers for pulmonary tuberculosis[J].PLoS One,2017,12(3):e0173304.
[5] 全国第五次结核病流行病学抽样调查技术指导组.2010年全国第五次结核病流行病学抽样调查报告[J].中国防痨杂志,2012,34(8):485-508. TECHNICAL GUIDANCE GROUP OF THE FIFTH NATIONAL TB EPIDEMIOLOGICAL SURVEY.The fifth national tuberculosis epidemiological survey in 2010[J].Chinese Journal of Antituberculosis,2012,34:485-508.(in Chinese)
[6] 王金果,刘爱林,周向梅,等.牛结核病诊断方法的研究进展[J].中国畜牧兽医,2010,37(7):194-198. WANG J G,LIU A L,ZHOU X M,et al.The advancement of diagnostic methods for bovine tuberculosis[J].China Animal Husbandry & Veterinary Medicine,2010,37(7):194-198.(in Chinese)
[7] MACK U,MIGLIORI G B,SESTER M,et al.LTBI:Latent tuberculosis infection or lasting immune responses to M.tuberculosis? A TBNET consensus statement[J].European Respiratory Journal,2009,33(5):956-973.
[8] TAKENAMI I,LOUREIRO C,JR M A,et al.Blood cells and interferon-gamma levels correlation in latent tuberculosis infection[J].Isrn Pulmonology,2013,2013:256148.
[9] WALZL G,RONACHER K,HANEKOM W,et al.Immunological biomarkers of tuberculosis[J].Nature Reviews Immunology,2011,11(5):343-354.
[10] SALGAME P,GEADAS C,COLLINS L,et al.Latent tuberculosis infection——Revisiting and revising concepts[J].Tuberculosis,2015,95(4):373-384.
[11] SUTHERLAND J S,DE JONG B C,JEFFRIES D J,et al.Production of TNF-α,IL-12(p40) and IL-17 can discriminate between active TB disease and latent infection in a West African cohort[J].PLoS One,2010,5(8):e12365.
[12] KHADER S A,BELL G K,PEARL J E,et al.IL-23 and IL-17 in the establishment of protective pulmonary CD4⁺ T cell responses after vaccination and during Mycobacterium tuberculosis challenge[J].Nature Immunology,2007,8(4):369-377.
[13] MATTOS A M M,ALMEIDA C D S,FRANKEN K L,et al.Increased IgG1,IFN-γ,TNF-α and IL-6 responses to Mycobacterium tuberculosis antigens in patients with tuberculosis are lower after chemotherapy[J].International Immunology,2010,22(9):775-782.
[14] GOOSEN W J,COOPER D,MILLER M A,et al.IP-10 is a sensitive biomarker of antigen recognition in whole-blood stimulation assays used for the diagnosis of Mycobacterium bovis infection in African buffaloes (Syncerus caffer)[J].Clinical and Vaccine Immunology,2015,22(8):974-978.
[15] RHODES S G,MCKINNA L C,STEINBACH S,et al.Use of antigen-specific interleukin-2 to differentiate between cattle vaccinated with Mycobacterium bovis BCG and cattle infected with M.bovis[J].Clinical and Vaccine Immunology,2014,21(1):39-45.
[16] XIN T,JIA H,DING J,et al.Assessment of a protein cocktail-based skin test for bovine tuberculosis in a double-blind field test in cattle[J].Clinical and Vaccine Immunology,2013,20(4):482-490.
[17] 高新桃.牛结核病诊断用细胞因子的筛选及其在临床检测中的初步应用[D].北京:中国农业科学院,2014. GAO X T.Screening of cytokines indicative of bovine TB and their preliminary application in clinical diagnosis[D].Beijing:Chinese Academy of Agricultural Sciences,2014.(in Chinese)
[18] WOOD P R,CORNER L A,PLACKETT P.Development of a simple,rapid in vitro cellular assay for bovine tuberculosis based on the production of gamma interferon[J].Research in Veterinary Science,1990,49(1):46-49.
[19] KHADER S A,COOPER A M.IL-23 and IL-17 in tuberculosis[J].Cytokine,2008,41(2):79-83.
[20] VORDERMEIER H M,VILLARREALRAMOS B,COCKLE P J,et al.Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis[J].Infection and Immunity,2009,77(8):3364-3373.
[21] TORRADO E,COOPER A M.IL-17 and Th17 cells in tuberculosis[J].Cytokine and Growth Factor Reviews,2010,21(6):455-462.
[22] ZHOU L,IVANOV I I,SPOLSKI R,et al.IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways[J].Cytokine,2007,39(1):967-974.
[23] OUYANG W,KOLLS J K,ZHENG Y.The biological functions of T helper 17 cell effector cytokines in inflammation[J].Immunity,2008,28(4):454-467.