摘要
目的:探索问号钩端螺旋体(简称钩体)对小鼠和人单核-巨噬细胞株NADPH氧化酶活性及活性氧簇( ROS)产生的影响,了解不同宿主巨噬细胞对问号钩体的杀菌机制。方法采用问号钩体56601株感染J774A.1和THP-1细胞建立钩体细胞感染模型,光度法测定细胞NADPH氧化酶活性和超氧离子O-2水平,免疫荧光法检测细胞ROS水平变化。结果光度法检测结果显示,问号钩体赖株感染2 h、4 h、12 h和24 h后,J774A.1细胞NADPH氧化酶活性分别从感染前的0.6190μmol·min-1· mg-1上升至0.3055μmol · min-1· mg-1、6.1415μmol · min-1· mg-1、1.4871μmol·min-1·mg-1和0.9646μmol · min-1· mg-1;THP-1细胞 NADPH 氧化酶活性分别从感染前0.7235μmol·min-1·mg-1上升至0.8842μmol·min-1·mg-1、1.8971 μmol·min-1·mg-1、1.1254μmol·min-1·mg-1和0.5627μmol·min-1·mg-1;超氧离子结果显示,J774A.1细胞超氧离子水平由正常细胞的0.1890 μmol/L 分别上调至0.2363μmol/L、0.2977μmol/L、0.3240μmol/L 和0.3057μmol/L;THP-1细胞由正常细胞的0.1237μmol/L 分别上调至0.1493μmol/L、0.2490μmol/L、0.2700μmol/L和0.2727μmol/L;免疫荧光检测结果表示,钩体感染J774A.1和THP-1细胞2 h、4 h、12 h和24 h后,细胞ROS水平较正常细胞逐渐变强,24 h出现降低。结论问号钩体感染J774A.1和THP-1细胞NADPH氧化酶活性和超氧离子O-2均显著上调,且J774A.1细胞上调更明显,结果有助于揭示巨噬细胞杀灭问号钩体的分子机制。
Objective To investigate the effects of Leptospira interrogans ( L. interrogans) infec-tion on the activities of NADPH oxidase ( nicotinamide adenine dinucleotide phosphate-oxidase) and the lev-els of reactive oxygen species (ROS) in THP-1 and J774A. 1 cells and to understand the bactericidal mecha-nisms of macrophages in different hosts against L. interrogans. Methods Human mononuclear macrophage cell line (THP-1 cells) and murine mononuclear macrophage cell line (J774A. 1 cells) were infected with L. interrogans strain 56601. The activities of NADPH oxidase and the levels of superoxide ion ( O-2 ) were measured with spectrophotography. Changes in the levels of ROS were detected with immunofluorescence as-say. Results Compared with the normal cells, the activities of NADPH oxidase in L. interrogans-infected J774A. 1 cells changed from 0. 619 0 μmol · min-1 · mg-1 to 0. 305 5 μmol · min-1 · mg-1 , 6. 141 5μmol·min-1 ·mg-1 , 1. 487 1μmol·min-1 ·mg-1 and 0. 964 6μmol·min-1 ·mg-1 after 2, 4, 12 and 24 hours of infection, respectively (P〈0. 05), while the activities of NADPH oxidase in L. interrogans-infected THP-1 cells were up-regulated from 0. 723 5μmol·min-1 ·mg-1 to 0. 884 2μmol·min-1 ·mg-1 , 1. 897 1μmol·min-1 ·mg-1 , 1. 125 4 μmol·min-1 ·mg-1 and 0. 562 7 μmol·min-1 ·mg-1 , respectively ( P〈0. 05). The levels of O-2 in L. interrogans-infected J774A. 1 cells at the time points of 2 h, 4 h, 12 h and 24 h after infection increased from 0. 189 0μmol/L to 0. 236 3μmol/L, 0. 297 7μmol/L, 0. 324 0μmol/L and 0. 305 7 μmol/L, respectively (P〈0. 05), while the levels of O-2 in L. interrogans-infected THP-1 cells rose from 0. 123 7 μmol/L to 0. 149 3 μmol/ L, 0. 249 0 μmol/ L, 0. 270 0 μmol/ L and 0. 272 7μmol/L, respectively (P〈0. 05). The fluorescence intensity of ROS in THP-1 and J774A. 1 cells increased gradually after infection with L. interrogans for 2 h and decreased after reaching the peak at 24 h. Conclu-sion Both the activities of NADPH oxidase and the levels of O-2 in J774A. 1 and THP-1 cells were signifi-cantly upregulated after infected with L. interrogans, especially in J774A. 1 cells. The results of this study provided references for further elucidating the bactericidal mechanisms of macrophages in different hosts against L. interrogans.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2016年第9期662-666,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金项目(81260250,81401679)
贵州省优秀青年科技人才培养对象专项资金项目(黔科合人字(2015)09号)
贵州省高层次创新型人才培养项目经费[黔科合(2016)4021]
贵州省传染病人才培养基地项目[黔人领发(2013)15号]
关键词
问号钩端螺旋体
巨噬细胞
杀菌机制
NADPH
ROS
Leptospira interrogans
Macrophage
NADPH
ROS
Bactericidal mechanism