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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 sustained infection model of Mycobacterium tuberculosis in mice and to study its immune response characteristics, female C57BL / 6 mice were selected and infected with Mycobacterium tuberculosis H37Rv via the tail vein and treated with isoniazid and rifampicin. The mice were sacrificed at 4, 8 and 12 weeks after infection, respectively. The count of lung and spleen of mice was counted by plate method. The levels of serum specific antibodies and subclasses were detected by enzyme linked immunosorbent assay (ELISA) The proportion of cells secreting cytokines after stimulation of splenic lymphocytes with M. tuberculosis antigen-purified protein derivative (PPD). The results showed that after 4 weeks of infection, the lg CFU of lung and spleen in mice reached 3.67 ± 0.25 and 3.54 ± 0.24, respectively, for at least 8 weeks. Drug treatment could effectively reduce the number of organism in the organ. After 12 weeks of infection, the serum levels of Mycobacterium tuberculosis-specific antibodies in the infected group were significantly increased (P <0.01), and IgG1 was the main group. The total IgG antibody level in the treated group was significantly lower than that in the infected group (P <0.01) And IgG2a is relatively high (P <0.05). The proportion of IFN-γ and IL-2 secreting cells in CD4 + splenic lymphocytes was significantly increased in infected group (P <0.01 and P <0.001), while the proportion of IL-4 secreting cells was significantly decreased P <0.01). The proportion of IL-2 and IL-4 secreting cells in the treatment group was significantly lower than that in the normal group (P <0.05 and P <0.01). The established model of persistent Mycobacterium tuberculosis infection in mice is expected to be used in the development and screening of therapeutic vaccines and drugs for tuberculosis.