结核分枝杆菌持续感染小鼠模型的建立及其免疫特征  

Establishment of a mouse model for Mycobacterium tuberculosis persistent infection and its immunological characteristics

作　　者：许承明[1] 康健[1] 王丽梅[1] 韩文东[2] 孙志平[2] 丁悦娜[2] 瞿涤[2,3] 柏银兰[1] 徐志凯[1] 

机构地区：[1]第四军医大学微生物学与病原生物学教研室,西安710032 [2]上海复旦大学三级生物安全防护实验室,上海200032 [3]复旦大学基础医学院教育部/卫生部医学分子病毒学重点实验室,上海200032

出　　处：《微生物与感染》2014年第2期71-76,共6页Journal of Microbes and Infections

基　　金："十二五"国家科技重大专项(2012ZX10003008);国家自然科学基金(81371774);"十二五"国家重大新药创制(2012ZX09301002)

摘　　要：为建立小鼠结核分枝杆菌持续感染模型并研究其免疫应答特征,选取雌性C57BL/6小鼠,经尾静脉感染结核分枝杆菌H37Rv株,并以异烟肼和利福平联合治疗。分别于感染后4、8、12周处死小鼠,用平板法计数肺和脾荷菌数,酶联免疫吸附试验(ELISA)检测血清中特异性抗体水平及亚类,流式细胞术检测CD4+脾淋巴细胞经结核分枝杆菌抗原——纯化蛋白衍生物(PPD)刺激后分泌细胞因子的细胞比例。结果显示,感染4周后小鼠肺和脾荷菌数lg CFU分别达3.67±0.25和3.54±0.24,至少持续8周;药物治疗可有效降低脏器荷菌数。感染12周后,感染组血清中抗结核分枝杆菌特异性抗体水平显著升高(P<0.01),且以IgG1为主;治疗组总IgG抗体水平显著低于感染组(P<0.01),且IgG2a相对较高(P<0.05)。感染组CD4+脾淋巴细胞中γ干扰素(IFN-γ)和白细胞介素2(IL-2)分泌细胞比例显著增加(P<0.01和P<0.001),而IL-4分泌细胞比例显著降低(P<0.01);治疗组IL-2和IL-4分泌细胞比例显著低于正常组(P<0.05和P<0.01)。本研究建立的小鼠结核分枝杆菌持续感染模型有望用于结核病治疗性疫苗和药物的研发及筛选。To establish a persistent infection mouse model and study its immunological characteristics ,female C57BL/6 mice were infected with Mycobacterium tuberculosis （M . tuberculosis） H37Rv at a dose of 1 ＆#215; 105 CFU/mouse by tail vein injection .Four weeks after infection ,the mice in the chemotherapy group were treated with isoniazid and rifampicin in drinking water for 8 weeks .At 4 ,8 and 12 weeks after infection ,6 mice from each study group were euthanized to examine bacterial burden in the lungs and spleens . The homogenized organs were serially diluted and plated on plates for bacterial count after 21-day incubation . Mouse sera were collected and the total anti-M . tuberculosis IgG and its subclasses were analyzed by enzyme-linked immunosorbent assay （ELISA ） . CD4^＋ splenocytes were cultured with purified protein＆amp;nbsp;derivative （PPD） stimulation ,and the percentages of interferon γ（IFN-γ） ,interleukin 2 （IL-2） ,IL-4 ,and tumor necrosis factor α （TNF-α） expressing cells were then identified by intracellular cytokine staining and flow cytometry .The results showed that the lgCFU of bacterial loads in lungs and spleens reached to 3 .67 ± 0 .25 and 3 .54 ± 0 .24 ,respectively 4 weeks after infection and the level retained for at least 8 weeks .The chemotherapy reduced the bacterial loads in organs significantly .12 weeks after infection ,M . tuberculosis specific total IgG level increased significantly in the untreated group （ P〈 0 .01 ） , especially IgG1 （ P〈0 .01） .The IgG level in the chemotherapy group also increased （P〈0 .05） but at a pace slower than that in the untreated group （P〈0 .01） .Compared with the untreated group ,the chemotherapy group had a lower IgG1 level （P〈0 .01） but a higher IgG2a level （P〈0 .05） .The untreated group had a significantly higher percentage of IFN-γ and IL-2 expressing CD4^＋ splenocytes （P〈0 .01 ,P〈0 .001） and lower percentage of IL-4 expressing CD4^＋ splenocytes compared with the contr

关 键 词：结核分枝杆菌 持续感染 小鼠模型 

分 类 号：R378.911[医药卫生—病原生物学] R446.5[医药卫生—基础医学]