白细胞黏附分子CD44在肺结核患者体内的表达水平、作用及机制研究
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摘要
目的:研究CD44在肺结核患者体内的表达,并初步探讨其可能的作用机制。方法:选取本院拟诊为肺结核患者236例,分为肺结核组(n=152)和非肺结核组(n=84),随机选取健康体检者100名作为健康对照组,qRT-PCR检测不同组别人群外周血单个核细胞PBMC和结核性胸膜炎患者胸水中CD44的表达,ELISA检测治疗前后结核性胸膜炎患者血浆和胸水中CD44的水平;进一步分析CD44在结核感染中的产生机制和发挥作用的方式;同时分析CD44与T-spot. TB联合检测对肺结核的诊断价值。结果:在肺结核患者外周血PBMC和结核性胸膜炎患者胸水中CD44 mRNA的表达水平显著高于非肺结核组和健康对照组,肺结核患者血浆和结核性胸膜炎患者胸水中CD44蛋白浓度显著高于非肺结核组和健康对照组;治疗后3、6、9月肺结核患者的外周血中CD44的mRNA水平,以及血浆中CD44浓度,显著降低,相比治疗前差异具有统计学意义;CD44和结核杆菌共作用的THP-1细胞可以显著提高细胞中CCL-2的表达,表明CD44通过促进结核杆菌感染的巨噬细胞中CCL2的表达来调节细胞的迁移;利用TNF-α中和抗体或者功能性TNF-α作用THP-1细胞,结果发现TNF-α可以显著提高细胞中CD44的表达。结论:CD44在结核患者体内高表达,可能是由于结核患者中高表达的TNF-α,从而刺激巨噬细胞产生CD44,为肺结核的临床诊断提供一定的研究基础。
Objective: To study the expression of CD44 in pulmonary tuberculosis patients and explore the possible mechanisms. Methods: 236 patients suspected with pulmonary tuberculosis were selected and divided into pulmonary tuberculosis group( n= 152) and non-pulmonary tuberculosis group( n= 84),and 100 healthy people were randomly selected as healthy control group. The expression of CD44 was evaluated by qRT-PCR and ELISA in peripheral blood and pleural effusion from different patients. The CD44 levels at pre and post-treatment time points were determined by ELISA. The inducing factors of increased CD44 and the potentialroles played by CD44 in the pathogenesis of TB were also evaluated. We elucidated whether CD44 detection could combine with T-spot. TB to diagnose pulmonary tuberculosis rapidly and accurately. Results: The expression of CD44 in pulmonary tuberculosis patients was higher compared with the non-pulmonary tuberculosis patients and healthy people,and would be down-regulated after treatment for 3,6 and 9 months. Besides,CD44 could not remove H37 Ra by the CFU assay,and could promote the expression of CCL-2,indicating that CCD4 promote the mobility the THP-1 via induction of CCL-2. Besides,TNF-α neutralizing antibody,added into the macrophages,could inhibit the expression of CD44,and functional TNF-α induced the expression of TNF-α. Conclusion: CD44 is highly expressed in pulmonary tuberculosis patients,which may be due to the high expression of TNF-α in pulmonary tuberculosis patients,stimulating macrophages to produce CD44,and it will provide a basis for clinical diagnosis of pulmonary tuberculosis.
Objective: To study the expression of CD44 in pulmonary tuberculosis patients and explore the possible mechanisms. Methods: 236 patients suspected with pulmonary tuberculosis were selected and divided into pulmonary tuberculosis group( n= 152) and non-pulmonary tuberculosis group( n= 84),and 100 healthy people were randomly selected as healthy control group. The expression of CD44 was evaluated by qRT-PCR and ELISA in peripheral blood and pleural effusion from different patients. The CD44 levels at pre and post-treatment time points were determined by ELISA. The inducing factors of increased CD44 and the potentialroles played by CD44 in the pathogenesis of TB were also evaluated. We elucidated whether CD44 detection could combine with T-spot. TB to diagnose pulmonary tuberculosis rapidly and accurately. Results: The expression of CD44 in pulmonary tuberculosis patients was higher compared with the non-pulmonary tuberculosis patients and healthy people,and would be down-regulated after treatment for 3,6 and 9 months. Besides,CD44 could not remove H37 Ra by the CFU assay,and could promote the expression of CCL-2,indicating that CCD4 promote the mobility the THP-1 via induction of CCL-2. Besides,TNF-α neutralizing antibody,added into the macrophages,could inhibit the expression of CD44,and functional TNF-α induced the expression of TNF-α. Conclusion: CD44 is highly expressed in pulmonary tuberculosis patients,which may be due to the high expression of TNF-α in pulmonary tuberculosis patients,stimulating macrophages to produce CD44,and it will provide a basis for clinical diagnosis of pulmonary tuberculosis.
引文
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[12]Giri PK,Verma I,Khuller GK.Adjunct immunotherapy with Ag85complex proteins based subunit vaccine in a murine model of Mycobacterium tuberculosis infection[J].Immunotherapy,2009,1(1):31-37.
[13]Li M,Yu DH,Cai H.The synthetic antimicrobial peptide KLKL5KLK enhances the protection and efficacy of the combined DNA vaccine against Mycobacterium tuberculosis[J].DNA Cell Biol,2008,27(8):405-413.
[14]Waters WR,Rahner TE,Palmer MV,et al.Expression of L-Selectin(CD62L),CD44,and CD25 on activated bovine T cells[J].Infect Immun,2003,71(1):317-326.
[15]Caruso AM,Serbina N,Klein E,et al.Mice deficient in CD4 Tcells have only transiently diminished levels of IFN-γ,yet succumb to tuberculosis[J].J lmmunol,1999,162(9):5407-1546.
[16]魏继英,史久利.国内结核性胸膜炎诊断和治疗现况[J].中国现代医生,2008,46(1):143-144.Wei JY,Shi JL.Current status of diagnosis and treatment of tuberculosis pleurisy in China[J].China Modern Doctor,2008,46(1):143-144.
[17]Le Y,Zhou Y,Iribarren P,et al.Chemokines and chemokine receptors:their mainfold in homeostasis and disease[J].Cell Mol Immunol,2004,1(2):95-104.
[18]Yuan GH,Masuko-Hongo K,Nishioka K.Role of Chemokines/Chemokine Receptor Systems in Cartilage Degradation[J].Drug News Perspect,2001,14(10):591-600.
[19]Ferrian S,Manca C,Lubbe S.A combination of baseline plasma immune markers can predict therapeutic response in multidrug resistant tuberculosis[J].PLoS One,2017,12(5):e176660.
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[22]Cooper AM,Magram J,Fenante J,et al.Interleukin 12(IL-12)is crucial to the development of protective immunity in mice intravenously infected with Mycobacterium tuberculosis[J].J Exp Med,1997,186(1):39-45.
[23]Palaniappan N,Anbalagan S,Narayanan S.Mitogen-activated protein kinases mediate Mycobacterium tuberculosis-induced CD44surface expression in monocytes[J].J Biosci,2012,37(1):41-54.
[24]孙翠芬,杨静华,董晓瑜,等.腹水中TB-AB、ADA和s CD44v6的检测及意义[J].现代肿瘤医学,2011,19(8):1608-1610.Sun CF,Yang JH,Dong XY,et al.Detection and significance of TB-AB,ADA and s CD44v6 in ascites[J].J Modern Oncol,2011,19(8):1608-1610.
[25]朱杰,王善菊,聂大平,等.DNA含量分析和非白细胞细胞群CD44分子的检测在胸腔积液诊断和鉴别诊断中的价值[J].大连医科大学学报,2009,31(2):200-203.Zhu J,Wang SJ,Nie DP,et al.Diagnostic value of DNA content and CD44 in non-leukocyte cells in pleural effusion[J].J Dalian Med Univ,2009,31(2):200-203.
[26]Widdison S,Siddiqui N,Easton V,et al.The bovine chemokine receptors and their mRNA abundance in mononuclear phagocytes[J].BMC Genomics,2010,11:439.
[27]Urata S,Izumi K,Hiratsuka K.CCL5 promotes migration of prostate cancer cells in the prostate cancer bone metastasis microenvironment[J].Cancer Sci,2017.[Ehead of print]
[28]Mirzadegan T,Diehl F,Ebi B,et al.Identification of the binding site for a novel class of CCR2b chemokine receptor antagonists binding to a common chemokine receptor motif within the helical bundle[J].J Biol Chem,2000,275(33):25562-25571.
[29]Kurihara T,Warr G,Loy J,et al.Defects in macrophage recruitment and host defense in mice lacking the CCR2chemokine receptor[J].J Exp Med,1997,186(10):1757-1762.
[30]Lee Y,Ryu JW,Chang H,et al.In vivo MR evaluation of the effect of the CCR2 antagonist on macrophage migration[J].Magn Reson Med,2010,64(1):72-79.
[31]Morrison H,Sherman LS,kgg J,et al.The NF2:umar suppressor gene product,merlin,mediates contact inhibition of growth through interactions with CD44[J].Genes Dev,2001,15(8):968-980.
[32]Tang J,Chen C,Zha C.Peripheral blood T cell TNF-αand IFN-γproduction stimulated by low molecular peptide of Mycobacterium tuberculosis heat-resistant antigen for differential diagnosis between pulmonary tuberculosis and latent tuberculosis infection[J].Nan Fang Yi Ke Da Xue Xue Bao,2017,37(11):1442-1447.
[33]李俊,缪苏宇,陈薇,等.肿瘤坏死因子-α对乳腺癌细胞CD44表达调控及其影响细胞迁徙机制的探讨[J].中华肿瘤防治杂志,2010,17(8):566-570.Li J,Miao SY,Chen W,et al.Study on the effect of TNF-αon the expression of CD44 in breast cancer cells and its mechanism of cell migration[J].Chin J Cancer Prevention Treatment,2010,17(8):566-570.
[34]Li XL,Xie N,Wang SW,et al.Diagnostic value of cerebrospinal fluid T-SPOT.TB for tuberculousis meningitis in china[J].Biomed Environ Sci,2017,30(9):681-684.
[35]Iqari H,Ishikawa S,Nakazawa T,et al.Lymphocyte subset analysis in Quanti FERON-TB Gold Plus and T-Spot.TB for latent tuberculosis infection in rheumatoid arthritis[J].J Infect Chemother,2018,24(2):110-116.
[36]Lee LN,Chou CH,Wang JY,et al.Enzyme-linked immunospot assay for interferon-gamma in the diagnosis of tuberculous pleurisy[J].Clin Mircrobiol Infect,2009,15(6):173-179.
[37]李红,唐神结,史祥,等.全血γ-干扰素释放试验对涂阴肺结核诊断价值的研究[J].中华临床医师杂志,2012,6(16):4894-4897.Li Y,Tang SJ,Shi X,et al.Study on the diagnostic value of whole blood IFN-γrelease test for smear-negative pulmonary tuberculosis[J].Chin J Clinicians,2012,6(16):4894-4897.
[38]霍霏霏,刘晓清.结核病免疫学诊断新进展-T-SPOT.TB[J].中华实验和临床感染病杂志,2010,4(2):202-205.Huo FF,Liu XQ.New progress in immunology diagnosis of tuberculosis-T-SPOT.TB[J].Chin J Exp Clin Infect Dis,2010,4(2):202-205.
[2]Jia Z,Cheng S,Ma Y.Tuberculosis burden in China:a high prevalence of pulmonary tuberculosis in household contacts with and without symptoms[J].BMC Infect Dis,2014,14:64.
[3]Amicosante M,Ciccozzi M,Markova R.Rational use of immunogiagnotic tools for tuberculosis infection:guidelines and cost effectiveness studies[J].New Microbiol,2010,33(2):93-107.
[4]陈效友.肺结核诊断的研究进展[J].中国临床医生,2013,41(3):11-14.Chen XY.Progress in diagnosis of pulmonary tuberculosis[J].JChin Physician,2013,41(3):11-14.
[5]Wu YB,Ye ZJ,Qin SM,et al.Combined detections of interleukin27,interferon-,and adenosine deaminase in pleural effusion for diagnosis of tuberculous pleurisy[J].Chin Med J(Engl),2013,126(17):3215-3221.
[6]Shamsuzzaman AK.Comparison between ELISA and ICT-MycoDot in adult pulmonary tuberculosis[J].Mymensingh Med J,2006,15(1):33-39.
[7]Cooper AM,Dalton DK,Stewart TA,et al.Disseminated tuberculosis in IFN-γgene-disrupted mice[J].J Exp Med,1993,178(6):2243-2247.
[8]Qi ZJ,Yu H,Zhang J,et al.Presepsin as a novel diagnostic biomarker for differentiating active pulmonary tuberculosis from bacterial community acquired pneumonia[J].Clin Chim Acta,2018,478(1):152-156.
[9]Lyadova I,Yeremeev V,Majorov K,et al.An ex vivo study of Tlymphocytes recovered from the lungs of I/St mice infected with and susceptible to Mycobacterium tuberculosis[J].Infect Immun,1998,66(10):4981-4988.
[10]Feng CG,Britton WJ,Palendira U,et al.Up-regulation of VCAM-1 and differential expansion of beta integrin-expressing T lymphocytes are associated with immunity to pulmonary Mycobacterium tuberculosis infection[J].J Immunol,2000,164(9):4853-4860.
[11]Leemans JC,Florquin S,Heikens M,et al.CD44 is a macrophage binding site for Mycobacterium tuberculosis that mediates macrophage recruitment and protective immunity against tuberculosis[J].J Clin Invest,2003,111(5):681-689.
[12]Giri PK,Verma I,Khuller GK.Adjunct immunotherapy with Ag85complex proteins based subunit vaccine in a murine model of Mycobacterium tuberculosis infection[J].Immunotherapy,2009,1(1):31-37.
[13]Li M,Yu DH,Cai H.The synthetic antimicrobial peptide KLKL5KLK enhances the protection and efficacy of the combined DNA vaccine against Mycobacterium tuberculosis[J].DNA Cell Biol,2008,27(8):405-413.
[14]Waters WR,Rahner TE,Palmer MV,et al.Expression of L-Selectin(CD62L),CD44,and CD25 on activated bovine T cells[J].Infect Immun,2003,71(1):317-326.
[15]Caruso AM,Serbina N,Klein E,et al.Mice deficient in CD4 Tcells have only transiently diminished levels of IFN-γ,yet succumb to tuberculosis[J].J lmmunol,1999,162(9):5407-1546.
[16]魏继英,史久利.国内结核性胸膜炎诊断和治疗现况[J].中国现代医生,2008,46(1):143-144.Wei JY,Shi JL.Current status of diagnosis and treatment of tuberculosis pleurisy in China[J].China Modern Doctor,2008,46(1):143-144.
[17]Le Y,Zhou Y,Iribarren P,et al.Chemokines and chemokine receptors:their mainfold in homeostasis and disease[J].Cell Mol Immunol,2004,1(2):95-104.
[18]Yuan GH,Masuko-Hongo K,Nishioka K.Role of Chemokines/Chemokine Receptor Systems in Cartilage Degradation[J].Drug News Perspect,2001,14(10):591-600.
[19]Ferrian S,Manca C,Lubbe S.A combination of baseline plasma immune markers can predict therapeutic response in multidrug resistant tuberculosis[J].PLoS One,2017,12(5):e176660.
[20]Tascon RE,Stavropoulos E,Lukacs KV,et a1.Protection against Mycobacterium tuberculosis infection by CD8 T cells requires production of gamma interferon[J].Infect Immun,1998,66(2):830-834.
[21]Vesosky B,Flaherty DK,Turner J.Thl cytokines facilitate CD8-T-cell-mediated early resistance to infection with Mycobacterium tuberculosis in old mice[J].Infect Immun,2006,74(6):3314-3324.
[22]Cooper AM,Magram J,Fenante J,et al.Interleukin 12(IL-12)is crucial to the development of protective immunity in mice intravenously infected with Mycobacterium tuberculosis[J].J Exp Med,1997,186(1):39-45.
[23]Palaniappan N,Anbalagan S,Narayanan S.Mitogen-activated protein kinases mediate Mycobacterium tuberculosis-induced CD44surface expression in monocytes[J].J Biosci,2012,37(1):41-54.
[24]孙翠芬,杨静华,董晓瑜,等.腹水中TB-AB、ADA和s CD44v6的检测及意义[J].现代肿瘤医学,2011,19(8):1608-1610.Sun CF,Yang JH,Dong XY,et al.Detection and significance of TB-AB,ADA and s CD44v6 in ascites[J].J Modern Oncol,2011,19(8):1608-1610.
[25]朱杰,王善菊,聂大平,等.DNA含量分析和非白细胞细胞群CD44分子的检测在胸腔积液诊断和鉴别诊断中的价值[J].大连医科大学学报,2009,31(2):200-203.Zhu J,Wang SJ,Nie DP,et al.Diagnostic value of DNA content and CD44 in non-leukocyte cells in pleural effusion[J].J Dalian Med Univ,2009,31(2):200-203.
[26]Widdison S,Siddiqui N,Easton V,et al.The bovine chemokine receptors and their mRNA abundance in mononuclear phagocytes[J].BMC Genomics,2010,11:439.
[27]Urata S,Izumi K,Hiratsuka K.CCL5 promotes migration of prostate cancer cells in the prostate cancer bone metastasis microenvironment[J].Cancer Sci,2017.[Ehead of print]
[28]Mirzadegan T,Diehl F,Ebi B,et al.Identification of the binding site for a novel class of CCR2b chemokine receptor antagonists binding to a common chemokine receptor motif within the helical bundle[J].J Biol Chem,2000,275(33):25562-25571.
[29]Kurihara T,Warr G,Loy J,et al.Defects in macrophage recruitment and host defense in mice lacking the CCR2chemokine receptor[J].J Exp Med,1997,186(10):1757-1762.
[30]Lee Y,Ryu JW,Chang H,et al.In vivo MR evaluation of the effect of the CCR2 antagonist on macrophage migration[J].Magn Reson Med,2010,64(1):72-79.
[31]Morrison H,Sherman LS,kgg J,et al.The NF2:umar suppressor gene product,merlin,mediates contact inhibition of growth through interactions with CD44[J].Genes Dev,2001,15(8):968-980.
[32]Tang J,Chen C,Zha C.Peripheral blood T cell TNF-αand IFN-γproduction stimulated by low molecular peptide of Mycobacterium tuberculosis heat-resistant antigen for differential diagnosis between pulmonary tuberculosis and latent tuberculosis infection[J].Nan Fang Yi Ke Da Xue Xue Bao,2017,37(11):1442-1447.
[33]李俊,缪苏宇,陈薇,等.肿瘤坏死因子-α对乳腺癌细胞CD44表达调控及其影响细胞迁徙机制的探讨[J].中华肿瘤防治杂志,2010,17(8):566-570.Li J,Miao SY,Chen W,et al.Study on the effect of TNF-αon the expression of CD44 in breast cancer cells and its mechanism of cell migration[J].Chin J Cancer Prevention Treatment,2010,17(8):566-570.
[34]Li XL,Xie N,Wang SW,et al.Diagnostic value of cerebrospinal fluid T-SPOT.TB for tuberculousis meningitis in china[J].Biomed Environ Sci,2017,30(9):681-684.
[35]Iqari H,Ishikawa S,Nakazawa T,et al.Lymphocyte subset analysis in Quanti FERON-TB Gold Plus and T-Spot.TB for latent tuberculosis infection in rheumatoid arthritis[J].J Infect Chemother,2018,24(2):110-116.
[36]Lee LN,Chou CH,Wang JY,et al.Enzyme-linked immunospot assay for interferon-gamma in the diagnosis of tuberculous pleurisy[J].Clin Mircrobiol Infect,2009,15(6):173-179.
[37]李红,唐神结,史祥,等.全血γ-干扰素释放试验对涂阴肺结核诊断价值的研究[J].中华临床医师杂志,2012,6(16):4894-4897.Li Y,Tang SJ,Shi X,et al.Study on the diagnostic value of whole blood IFN-γrelease test for smear-negative pulmonary tuberculosis[J].Chin J Clinicians,2012,6(16):4894-4897.
[38]霍霏霏,刘晓清.结核病免疫学诊断新进展-T-SPOT.TB[J].中华实验和临床感染病杂志,2010,4(2):202-205.Huo FF,Liu XQ.New progress in immunology diagnosis of tuberculosis-T-SPOT.TB[J].Chin J Exp Clin Infect Dis,2010,4(2):202-205.