常用实验小鼠感染诺如病毒后外周血免疫指标分析
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摘要
目的通过小鼠感染MNV后外周血免疫指标的改变来评估MNV感染对小鼠免疫功能的影响。方法选取KM,BALB/c,BALB/c-nu,C57BL/6及NIH等五个品系小鼠,每个品系分成MNV感染组和对照组,于感染前(第0天)和感染后的第7、14、21、28天从眼周静脉采集抗凝血,流式细胞术测定总T细胞、CD4~+ T细胞、CD8~+ T细胞、总B细胞、NK细胞及NK-T细胞占总淋巴细胞的百分比及干扰素、白介素、趋化因子等13种细胞因子的含量,比较感染后各指标总体均值的差异,分析各时间点变化情况。结果与对照组相比,KM小鼠感染组总T细胞、CD4~+ T细胞含量显著增高(P<0.01),CD4/CD8显著增高(P<0.05),总B细胞及CXCL10含量显著降低(P<0.01);BALB/c小鼠CD8~+ T细胞显著降低(P<0.05),IFN-γ含量显著增高(P<0.01);BALB/c-nu小鼠各免疫细胞均无统计学差异,TNF-α、CXCL1及CXCL10显著增高(P<0.01),CCL2显著增高(P<0.05),IFN-β显著降低(P<0.05);C57BL/6小鼠CD4~+(P<0.05)CD8~+ T细胞(P<0.01)均显著降低,总B细胞及IL-10显著增高(P<0.05),CCL5及GM-CSF显著降低(P<0.05);NIH小鼠总T细胞和CD8~+ T细胞显著增高(P<0.01),CD4/CD8显著降低(P<0.05),总B细胞显著降低(P<0.01),细胞因子均无统计学差异。对于有统计学差异的免疫指标,有半数感染第7天即出现显著差异,大多数第28天仍存在显著差异。结论 MNV的感染会影响正常小鼠的细胞免疫和体液免疫应答,而且不同品系的动物影响不同,建议在进行免疫相关的动物实验时选择MNV阴性小鼠,以避免实验结果出现偏差。
Objective To evaluate the effect of murine norovirus(MNV) infection on immune function in mice by assessing the changes in peripheral blood immune parameters. Methods KM, BALB/c, BALB/c-nu, C57 BL/6, and NIH mice were selected. Each strain was divided into an MNV infection group and a control group. Anticoagulated blood samples were collected from the periocular vein plexus before infection(day 0) and at 7, 14, 21, and 28 d post-infection. Flow cytometry was used to determine the percentage of total T cells, CD4~+ T cells, CD8~+ T cells, total B cells, NK cells and NK-T cells in the total lymphocytes and the levels of 13 cytokines such as interferon and interleukin, and chemokines. To compare the differences in the mean values of each index after infection, the changes at each time point were analyzed. Results Compared with the control group, the level of total T cells and CD4~+ T cells in the infected group of KM mice significantly increased(P<0.01), CD4/CD8 significantly increased(P<0.05), and the total B cell and CXCL10 content significantly decreased(P<0.01). The level of CD8~+ T cells in infected BALB/c mice significantly decreased(P<0.05), and IFN-γ levels significantly increased(P<0.01). There was no significant difference between the levels of immune cells in infected and control BALB/c-nu mice, although in the infected BALB/c-nu mice TNF-α, CXCL1 and CXCL10 significantly increased(P<0.01), CCL2 significantly increased(P<0.05), and IFN-β significantly decreased(P<0.05). CD4~+(P<0.05) and CD8~+ T cells(P<0.01) in the infected C57 BL/6 mice significantly decreased, total B cells and IL-10 significantly increased(P<0.05), and CCL5 and GM-CSF significantly decreased(P<0.05). Total T cells and CD8~+ T cells in the infected NIH mice significantly increased(P<0.01), CD4/CD8 significantly decreased(P<0.05), and total B cells significantly decreased(P<0.01), while there were no significant differences in cytokine levels. In terms of statistically significant changes in immune indicators, half of all immune indicators showed significant differences on the 7 th day of infection, and most still demonstrated significant differences on the 28 th day. Conclusions MNV infection affects cellular and humoral immune responses in otherwise healthy mice, and different mouse strains demonstrate different effects. Researchers should select MNV-negative mice in immunologically relevant animal experiments to avoid bias in the experimental results.
Objective To evaluate the effect of murine norovirus(MNV) infection on immune function in mice by assessing the changes in peripheral blood immune parameters. Methods KM, BALB/c, BALB/c-nu, C57 BL/6, and NIH mice were selected. Each strain was divided into an MNV infection group and a control group. Anticoagulated blood samples were collected from the periocular vein plexus before infection(day 0) and at 7, 14, 21, and 28 d post-infection. Flow cytometry was used to determine the percentage of total T cells, CD4~+ T cells, CD8~+ T cells, total B cells, NK cells and NK-T cells in the total lymphocytes and the levels of 13 cytokines such as interferon and interleukin, and chemokines. To compare the differences in the mean values of each index after infection, the changes at each time point were analyzed. Results Compared with the control group, the level of total T cells and CD4~+ T cells in the infected group of KM mice significantly increased(P<0.01), CD4/CD8 significantly increased(P<0.05), and the total B cell and CXCL10 content significantly decreased(P<0.01). The level of CD8~+ T cells in infected BALB/c mice significantly decreased(P<0.05), and IFN-γ levels significantly increased(P<0.01). There was no significant difference between the levels of immune cells in infected and control BALB/c-nu mice, although in the infected BALB/c-nu mice TNF-α, CXCL1 and CXCL10 significantly increased(P<0.01), CCL2 significantly increased(P<0.05), and IFN-β significantly decreased(P<0.05). CD4~+(P<0.05) and CD8~+ T cells(P<0.01) in the infected C57 BL/6 mice significantly decreased, total B cells and IL-10 significantly increased(P<0.05), and CCL5 and GM-CSF significantly decreased(P<0.05). Total T cells and CD8~+ T cells in the infected NIH mice significantly increased(P<0.01), CD4/CD8 significantly decreased(P<0.05), and total B cells significantly decreased(P<0.01), while there were no significant differences in cytokine levels. In terms of statistically significant changes in immune indicators, half of all immune indicators showed significant differences on the 7 th day of infection, and most still demonstrated significant differences on the 28 th day. Conclusions MNV infection affects cellular and humoral immune responses in otherwise healthy mice, and different mouse strains demonstrate different effects. Researchers should select MNV-negative mice in immunologically relevant animal experiments to avoid bias in the experimental results.
引文
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[14] 陈功义,银岭,肖传斌.板蓝根多糖对缺乳仔鼠免疫器官发育及T淋巴细胞亚群的影响[J].中兽医医药杂志,2015(1):15-18.
[15] 孙晓雨,崔子寅,张明亮,等.枸杞多糖和茯苓多糖对免疫抑制小鼠免疫增强及对肠道黏膜的免疫调节作用[J].中国兽医学报,2015,35(3):450-455.
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[20] 宋玉龙,丘富安,吴秀钦,等.硒化修饰太子参多糖对免疫损伤小鼠的免疫保护作用[J].中国兽医学报,2017,37(11):2163-2167.
[21] 闫志强,郑华,付文贵,等.女黄颗粒对免疫抑制小鼠外周血T淋巴细胞亚群及脾脏细胞因子mRNA表达的影响[J].南方农业学报,2018,49(10):2070-2076.
[22] He JH,Zhang R,Xie LJ,et al.The clinical significance of tumor necrosis factor-α level in anti-neutrophil cytoplasmic antibody-associated vasculitis[J].Immunol J,2017,33(12):1060-1065.
[23] 吴凤霞,袁国华.趋化因子及其受体研究进展[J].川北医学院学报,2008,23(3):297-300.
[24] 李臻,信珊珊,董晓磊,等.萝卜硫素对免疫抑制小鼠免疫功能的影响[J].济宁医学院学报,2017,40(6):393-397.
[25] Niendorf S,Klemm U,Mas Marques A,et al.Infection with the persistent murine norovirus strain MNV-S99 suppresses IFN-β release and activation of Stat1 in vitro[J].PLoS One,2016,11(6):e0156898.
[26] Hsu CC,Piotrowski SL,Meeker SM,et al.Histologic lesions induced by murine norovirus infection in laboratory mice[J].Vet Pathol,2016,53(4):754-763.
[2] 王翠娥,陈立超,周倩,等.实验大鼠和小鼠多种病毒的血清学检测结果分析[J].实验动物科学,2014,31(2):20-24.
[3] Hsu CC,Riley LK,Wills HM,et al.Persistent infection with and serologic cross-reactivity of three novel murine noroviruses[J].Comp Med,2006,56:247-251.
[4] Mumphrey SM,Changotra H,Moore TN,et al.Murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts,but clinical disease is prevented by STAT1-dependent interferon responses[J].J Virol,2007,81(7):3251-3263.
[5] Arias A,Bailey D,Chaudhry Y,et al.Development of a reverse-genetics system for murine norovirus 3:long-term persistence occurs in the caecum and colon[J].J Gen Virol,2012,93(Pt 7):1432-1441.
[6] Nice TJ,Strong DW,McCune BT,et al.A single-amino-acid change in murine norovirus NS1/2 is sufficient for colonic tropism and persistence[J].J Virol,2013,87(1):327-334.
[7] Shortland A,Chettle J,Archer J,et al.Pathology caused by persistent murine norovirus infection[J].J Gen Virol,2014,95(2):413-422.
[8] Thackray LB,Wobus CE,Chachu KA,et al.Murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence[J].J Virol,2007,81(19):10460-10473.
[9] 李晓波,付瑞,岳秉飞,等.鼠诺如病毒的体外分离与鉴定[J].中国病毒病杂志,2011,1(4):293-296.
[10] 李晓波,付瑞,王吉,等.鼠诺如病毒分离株全基因组序列的测定及分析[J].中国病毒病杂志,2013,3(5):335-342.
[11] 梁爽,左之才,辜旭初,等.肥胖对非致死性肺炎模型小鼠脾脏、外周血T淋巴细胞亚群的影响[J].免疫学杂志,2019,35(1):21-28.
[12] Karst SM,Wobus CE,Lay M,et al.STAT1-dependent innate immunity to a Norwalk-like virus[J].Science,2003,299(5612):1575-1578.
[13] Guo L,Wang JW,Zhou HL,et al.Intranasal administration of a recombinant adenovirus expressing the norovirus capsid protein stimulates specific humoral,mucosal,and cellular immune responses in mice[J].Vaccine,2008,26:460-468.
[14] 陈功义,银岭,肖传斌.板蓝根多糖对缺乳仔鼠免疫器官发育及T淋巴细胞亚群的影响[J].中兽医医药杂志,2015(1):15-18.
[15] 孙晓雨,崔子寅,张明亮,等.枸杞多糖和茯苓多糖对免疫抑制小鼠免疫增强及对肠道黏膜的免疫调节作用[J].中国兽医学报,2015,35(3):450-455.
[16] Changotra H,Jia Y,Moore TN,et al.Type I and type II interferons inhibit the translation of murine norovirus proteins[J].J Virol,2009,83(11):5683-5692.
[17] Maloney NS,Thackray LB,Goel G,et al.Essential cell-autonomous role for interferon (IFN) regulatory factor 1 in IFN-gamma-mediated inhibition of norovirus replication in macrophages[J].J Virol,2012,86(23):12655-12664.
[18] Rodriguez MR,Monte K,Thackray LB,et al.ISG15 functions as an interferon-mediated antiviral effector early in the murine norovirus life cycle[J].J Virol,2014,88(16):9277-9286.
[19] 谭晓宇,杨勇,王科斯,等.灵芝红景天复方制剂对小鼠免疫功能的影响[J].免疫学杂志,2019,35(1):59-64.
[20] 宋玉龙,丘富安,吴秀钦,等.硒化修饰太子参多糖对免疫损伤小鼠的免疫保护作用[J].中国兽医学报,2017,37(11):2163-2167.
[21] 闫志强,郑华,付文贵,等.女黄颗粒对免疫抑制小鼠外周血T淋巴细胞亚群及脾脏细胞因子mRNA表达的影响[J].南方农业学报,2018,49(10):2070-2076.
[22] He JH,Zhang R,Xie LJ,et al.The clinical significance of tumor necrosis factor-α level in anti-neutrophil cytoplasmic antibody-associated vasculitis[J].Immunol J,2017,33(12):1060-1065.
[23] 吴凤霞,袁国华.趋化因子及其受体研究进展[J].川北医学院学报,2008,23(3):297-300.
[24] 李臻,信珊珊,董晓磊,等.萝卜硫素对免疫抑制小鼠免疫功能的影响[J].济宁医学院学报,2017,40(6):393-397.
[25] Niendorf S,Klemm U,Mas Marques A,et al.Infection with the persistent murine norovirus strain MNV-S99 suppresses IFN-β release and activation of Stat1 in vitro[J].PLoS One,2016,11(6):e0156898.
[26] Hsu CC,Piotrowski SL,Meeker SM,et al.Histologic lesions induced by murine norovirus infection in laboratory mice[J].Vet Pathol,2016,53(4):754-763.