羊口疮病毒ORFV129蛋白对树突状细胞成熟和功能的影响
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摘要
为了探讨羊口疮病毒ORFV129蛋白对小鼠骨髓树突状细胞(DCs)生物免疫学功能的影响,从小鼠骨髓腔中分离获得单核细胞,诱导分化为未成熟DCs并用不同浓度的ORFV129蛋白进行刺激,采用流式细胞术检测成熟DCs的表面标记物MHC-Ⅱ、CD40等表达及吞噬FITC-dextran的能力,以及检测ORFV129蛋白作用DCs后分泌IFN-γ、IL-6等细胞因子的水平;采用CCK8法检测ORFV129蛋白对DCs刺激T细胞增殖的影响以及检测培养上清中IFN-γ、IL-5的水平。结果显示,经10μg与5μg浓度的ORFV129蛋白作用24 h后,DCs表面分子CD80、CD40的表达显著上调,与未处理组相比差异显著(P<0.05);经ORFV129蛋白刺激后DCs吞噬FITC-dextran的能力下降;10μg的ORFV129蛋白极显著地促进了IFN-γ、IL-6、IL-12p40等细胞因子的表达,与空白组相比差异极显著(P<0.01);另外,经ORFV129蛋白处理的DCs还可刺激T细胞增殖,以及使培养上清中IFN-γ的分泌量显著增加。ORFV129蛋白可上调DCs表面MHC-Ⅱ、CD40、CD80和CD86分子表达,降低其吞噬能力,促进DCs分泌IFN-γ、IL-6、IL-10、IL-12p40等细胞因子,还可刺激T细胞增殖,表明ORFV129蛋白对DCs的成熟起到促进作用。
To investigate the biological and immunological functions of dendritic cells(DCs) in mice infected by ORFV129,mononuclear cells were isolated from the bone marrow of mice and induced into immature DCs by addition of rm-CSF and rm IL-4.The im DC were stimulated by different concentrations of the ORFV129 protein,the expression of MHC-Ⅱ and CD40 surface markers of mature DCs and the ability to phagocytosis FITC-dextran were determined by the flow cytometry assay,and the cytokines such as IFN-γand IL-6 were detected by the indirect ELISA.At the same time the effect of DCs on the proliferation of T cells was determined by CCK8.The expression of CD40 and CD80 on the surface of DCs was significantly up-regulated after 24 h treatment with different concentrations of ORFV129 protein,P<0.01 compared with the blank control.The ability of uptaking FITC-dextran was lower than before and the concentration of ORFV129 protein at 10 μg significantly promoted the expression of cytokines such as IFN-γ,IL-6 and IL-12 p40,compared with the blank control(P<0.01).In addition,DCs treated with ORFV129 protein was able to stimulate T cell proliferation,too and the secretion of IFN-γ was significantly increased in the culture supernatant.ORFV129 protein was able to up-regulate the expression of MHC-Ⅱ CD40,CD80 and CD86 molecules on DCs and reduce their phagocytosis ability,promote secretion of IFN-γ,IL-6,IL-10,IL-12 p40 and so on.ORFV129 protein was able to stimulate T cell proliferation,too.In conclusion,ORFV129 can promote the maturation of DCs.
To investigate the biological and immunological functions of dendritic cells(DCs) in mice infected by ORFV129,mononuclear cells were isolated from the bone marrow of mice and induced into immature DCs by addition of rm-CSF and rm IL-4.The im DC were stimulated by different concentrations of the ORFV129 protein,the expression of MHC-Ⅱ and CD40 surface markers of mature DCs and the ability to phagocytosis FITC-dextran were determined by the flow cytometry assay,and the cytokines such as IFN-γand IL-6 were detected by the indirect ELISA.At the same time the effect of DCs on the proliferation of T cells was determined by CCK8.The expression of CD40 and CD80 on the surface of DCs was significantly up-regulated after 24 h treatment with different concentrations of ORFV129 protein,P<0.01 compared with the blank control.The ability of uptaking FITC-dextran was lower than before and the concentration of ORFV129 protein at 10 μg significantly promoted the expression of cytokines such as IFN-γ,IL-6 and IL-12 p40,compared with the blank control(P<0.01).In addition,DCs treated with ORFV129 protein was able to stimulate T cell proliferation,too and the secretion of IFN-γ was significantly increased in the culture supernatant.ORFV129 protein was able to up-regulate the expression of MHC-Ⅱ CD40,CD80 and CD86 molecules on DCs and reduce their phagocytosis ability,promote secretion of IFN-γ,IL-6,IL-10,IL-12 p40 and so on.ORFV129 protein was able to stimulate T cell proliferation,too.In conclusion,ORFV129 can promote the maturation of DCs.
引文
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[19]ZHANG X,TARNBACH M N.An excess of the proinflammatory cytokines IFN-γand IL-12 impairs the development of the memory CD8+T cell response to chlamydia trachomatis[J].Journal of Immunology,2015,195(4):1665-1675.
[20]ESPINOSA J R,SAMY K P,KIRK A D.Memory T cells in organ transplantation:progress and challenges[J].Nature Reviews Nephrology,2016,12(6):339-347.
[2]HOSA MANI M,SCAGLIARINI A,BHANUPRAKASH V,et al.Orf:an update on current research and future perspectives[J].Expert Review of Anti-Infective Therapy,2009,7(7):879-893.
[3]吉艳红,尚佑军,王光祥,等.羊口疮病毒F1L基因的克隆及其B细胞表位预测[J].中国兽医科学,2010,40(11):1101-1105.JI Yan-hong,SHANG You-jun,WANG Guang-xiang,et al.Cloning and prediction of B cell epitopes for F1L gene of Orf virus[J].Chinese Veterinary Science,2010,40(11):1101-1105.(in Chinese)
[4]CHEN H,WEI L,KUANG Z,et al.The whole genomic analysis of Orf virus strain HN3/12 isolated from Henan Province,central China[J].Bmc Veterinary Research,2017,13(1):260-271.
[5]MCELROY M C,BASSETT H F.The development of oral lesions in lambs naturally infected with orf virus[J].Veterinary Journal,2007,174(3):663-664.
[6]ZHAO K,SONG D,HE W,et al.Identification and phylogenetic analysis of an Orf virus isolated from an outbreak in sheep in the Jilin Province of China[J].Veterinary Microbiology,2010,142(3-4):408-415.
[7]VENKATESAN G,BALAMURΜGAN V,BORA D P,et al.Sequence and phylogenetic analyses of an Indian isolate of orf virus from sheep[J].Veterinaria Italiana,2011,47(3):323-342.
[8]STEINMAN R M.Dendritic cells and the control of immunity:enhancing the efficiency of antigen presentation[J].The Mount Sinai Journal of Medicine,New York,2001,68(3):160-166.
[9]GIANOTTI L,SARGENTI M,GALBIATI F,et al.Phenotype and function of dendritic cells and T-lymphocyte polarization in the human colonic mucosa and adenocarcinoma[J].European Journal of Surgical Oncology the Journal of the European Society of Surgical Oncology&the British Association of Surgical Oncology,2008,34(8):883-889.
[10]AGRAWAL A,SRIDHARAN A,PRAKASH S,et al.Dendritic cells and aging:consequences for autoimmunity[J].Expert Review of Clinical Immunology,2012,8(1):73-80.
[11]LI X,HAN Y,ZHOU Q,et al.Apigenin,a potent suppressor of dendritic cell maturation and migration,protects against collagen-induced arthritis[J].Journal of Cellular and Molecular Medicine,2016,20(1):170-180.
[12]MOHAMED M R,RAHMAN M M,RICE A,et al.Cowpox virus expresses a novel ankyrin repeat NF-kappab inhibitor that controls inflammatory cell influx into virus-infected tissues and is critical for virus pathogenesis[J].Journal of Virology,2009,83(18):9223-9236.
[13]BLOY N,POL J,ARANDA F,et al.Trial watch:Dendritic cell-based anticancer therapy[J].Oncoimmunology,2014,3(11):e963424.
[14]LIN Y L,HU Y C,LIANG C C,et al.Enterovirus-71virus-like particles induce the activation and maturation of human monocyte-derived dendritic cells through TLR4 signaling[J].Plos One,2014,9(10):e111496.
[15]CELLA M,SALLUSTO F,LANZAVECCHIA A.Origin,maturation and antigen presenting function of dendritic cells[J].Current Opinion in Immunology,1997,9(1):10-16.
[16]WAN H,DUPASQUIER M.Dendritic cells in vivo and in vitro[J].Cellular&Molecular Immunology,2005,2(1):28-35.
[17]朱诗语,王春凤,杨桂连.树突状细胞在病毒感染动物免疫应答中的作用[J].畜牧与兽医,2014,46(12):106-108.ZHU Shi-yu,WANG Chun-feng,YANG Gui-lian.The role of dendritic cells in the immune response of virus-infected animals[J].Animal Husbandry﹠Veterinary Medicine,2014,46(12):106-108.(in Chinese)
[18]WANG X,EATON M,MAYER M,et al.Porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability[J].Archives of Virology,2007,152(2):289-303.
[19]ZHANG X,TARNBACH M N.An excess of the proinflammatory cytokines IFN-γand IL-12 impairs the development of the memory CD8+T cell response to chlamydia trachomatis[J].Journal of Immunology,2015,195(4):1665-1675.
[20]ESPINOSA J R,SAMY K P,KIRK A D.Memory T cells in organ transplantation:progress and challenges[J].Nature Reviews Nephrology,2016,12(6):339-347.