分枝杆菌持续感染小鼠模型的建立及其特征分析

Establishment of mouse models of persistent tuberculosis and characteristics of that infection

目的建立不同毒力的分枝杆菌持续感染小鼠模型并分析其免疫应答特征,为结核病新型疫苗及药物评价研究奠定基础。方法分别用结核分枝杆菌(Mtb)标准毒株H37Rv、减毒株H37Ra及疫苗株BCG经尾静脉注射感染BALB/c小鼠,5×10~4 CFU/只。感染后4、8周观察小鼠一般状态及体重变化;观察脾脏大体情况和肺组织病理改变;检测分枝杆菌特异性抗体水平、脾淋巴细胞刺激指数(SI)及Th1/Th2细胞因子分泌情况;采用平板计数法计脾、肺脏荷菌数CFUs。结果不同毒力分枝杆菌感染8周后,各组小鼠体重无显著差异;H37Rv感染组脾脏体积显著增大,肺组织病理切片显示菌株感染可引起不同程度的病理损伤;感染4周后小鼠血清特异性抗体水平升高,且感染后8周抗体水平高于4周水平,但不同菌株感染组间差异无统计学意义;不同毒力分枝杆菌感染小鼠的脾淋巴细胞刺激指数(SI)均升高,以H37Rv和H37Ra感染组脾淋巴细胞增殖更显著。H37Rv感染4周后IFN-γ、IL-10分泌水平显著升高(P<0.05)。感染4周后,H37Rv小鼠脾脏荷菌数Log_(10)CFU显著高于BCG组,为4.389±0.1245,而H37Ra感染组与BCG组无显著差异,为4.068±0.2184;各感染组肺荷菌数Log_(10)CFU无显著差异;感染8周后,H37Ra、H37Rv感染组小鼠脾脏荷菌数显著高于BCG组(P<0.05),而肺部荷菌数组间无差异。小鼠呈现持续感染状态。结论本研究成功建立了不同毒力分枝杆菌持续感染小鼠模型,该模型可用于结核病疫苗及药物的研发及筛选。

Objectives To establish a mouse model of persistent tuberculosis involving Mycobacterium tuberculosis with different levels of virulence and to study the characteristics of that infection in order to lay a new foundation for the assessment and study of new vaccines and drugs against tuberculosis. Methods Female BALB/e mice were intravenously infected via the tail vein with 5 × 10^4 colony-forming units （CFUs） of the H37Rv standard virulent strain, the H37Ra strain, or the BCG vaccine strain of M. tuberculosis. Four and 8 weeks after infection, changes in the general status of mice and their weight were observed. Samples of lung and spleen tissues from infected mice were collected for histopathologic examination after HE staining. Mouse sera were tested for the titer of antibodies against M. tuberculosis using ELISA. Splenic lymphocytes of infected mice were isolated for an MTS assay o{ lymphocyte proliferation, and Th1/Th2 cytokines were detected with ELISA. The bacterial load in the spleen and lungs was determined by counting colony-forming units （CFUs） on 7H10 plates. Results There were no significant differences in the weight of mice 8 weeks after infection with strains that had differing levels of virulence. Macroscopic splenomegaly was observed in mice infected with H37Rv. Pathological lesions occurred in all infected mice according to histopathological slices. Four weeks after infection, Mycobacterium-specific antibodies were detected, and the antibody titer in infected mice did not differ significantly. The proliferation of spleen lymphocytes and the production of Th1/Th2 cytokines increased significantly in mice infected with H37Ra and mice infected with H37Rv. Four weeks after infection, the production of IFN-γ and IL-10 increased significantly in mice infected with H37Rv （P〈0.05）. The bacterial load in the spleen of mice infected with H37Rv increased to 4. 389~0. 1245 log10 CFU, which was significantly higher than the load in mice infected with BCG （P〈0.05）. The bacterial load in mice infected with H37Ra was 4. 068± 0. 2184 log10 CFU, but the load did not differ significantly between mice infected with H37Ra and mice infected with BCG. There were no differences in the bacterial load in the lungs of the 3 infected groups. Eight weeks after infection, the bacterial load in the spleen of mice infected with H37Ra and mice in- fected with H37Rv increased significantly in comparison to that in mice infected with BCG （P〈0. 05）, but the bacterial load in the lungs did not differ among the 3 groups. The bacterial load in organs remained the same for at least 8 weeks, indicating a persistent infection. Conclusion A mouse model of persistent tuberculosis was successfully established and the characteristics of that infection were studied. This work will provide a useful animal model for further research and development of new vaccines and drugs against tuberculosis.