| Objectives: 1. To investigate the cellular and humoral immune responses in mice induced by Mycobacterium tuberculosis H37Ra. 2. To evaluate the protective efficacy against a virulent Mycobacterium tuberculosis strain H37Rv after immunization with Mycobacterium tuberculosis H37Ra in mice.Methods: 1. BALB/c mice were divided into NS, BCG and H37Ra group randomly, and intracutaneously injected at caudal region with NS, BCG and H37Ra respectively. After 6 weeks of immunization, the delayed type hypersensitivity reaction was observed. 2. At 8 weeks after immunization, some mice were sacrificed, then the organ weight indexes were determined. The histopathological changes of lungs were analyzed. 3. The total serum IgG was detected by ELISA. 4. The percentage of CD3+T, CD4+T and CD8+T- lymphocyte in spleen was detected by flow cytometry, and the ratio of CD4+T/CD8+T was also determined. 5. The spleen lymphocyte of mice was cultured with PPD in vitro, the proliferation of splenocyte was detected by MTT assay. The product of IFN-γand IL-4 in the supernatants of culture was determined by ELISA. 6. After 8 weeks of immunization, BALB/c mice were challenged via intraperitoneal route with MTB H37Rv strain. Four weeks after challenge, mice were sacrificed, then the organ weight indexes were determined. 7. The lung,liver and spleen tissues were harvested to quantitate the viable mycobacteria. 8. And the lung tissue was also harvested to have the histopathological examination.Results: 1. The delayed-type hypersensitivity reaction could be induced in mice after immunization with H37Ra, 24h after injection was the most optimal observing time. 2. After 8 weeks of immunization,the weight indexes of liver,lung,spleen and kidney were similar among the vaccinated groups. The histopathological changes of lungs were appeared no obvious difference. 3. The percentage of CD3+T-lymphocyte in mice of H37Ra vaccination was significantly higher than that of the NS control subjects (41.63±1.68)% vs (38.44±1.87)% (P<0.05). Compared with the BCG group, the percentage of CD3+T,CD4+T and CD8+T-lymphocyte in mice of H37Ra vaccination were slightly higher, and the percentage of CD4+T,CD8+T lymphocyte and the ratio of CD4+T/CD8+T had no significant difference among 3 groups (P>0.05). 4. The total serum antibody IgG titer, the proliferation of antigen- specific lymphocyte, the product of IFN-γand IL-4 in the H37Ra group were significantly higher than those of the NS group (P<0.05). 5. At 4 weeks after challenge, compared with the NS group, weight indexes of lung, spleen, kidney and liver of the BCG and H37Ra groups were significantly decreased (P<0.05), while there was no significant difference between the H37Ra and BCG groups. 6. The spleen,liver and lung of mice were isolated, dissociated and suitable dilutions of the homogenates were plated on Lowenstein-Jensen culture. The numbers of CFU were counted 21 days after incubation. Compared with the NS group, the mean numbers of viable bacteria in the lungs,spleens,livers of the H37Ra group reduced 0.954,1.228 and 0.883 log10CFU respectively (P<0.05). The viable bacteria in the lungs,spleens,livers were no difference between the H37Ra and BCG groups. 7. Histopathology of the lungs showed: there were a little of hemorrhagic and serous effusion in alveolar space of the H37Ra group, and alveolar septa was thickened along with the massive infiltration of macrophages, some granulomas were appeared. The structure of alveoli of the BCG group was integrity, some macrophages were infiltrated, alveoli wall was a little thickened. The pathological changes of the NS subjects were extensive and severity. There was much serous and hemorrhagic effusion in alveolar space. Most of alveoli wall was thickened. The structure of alveoli was partly destroyed.Conclusions: 1. Mycobacterium tuberculosis H37Ra could induce specific cellular and humoral immune responses in mice; 2. Protective efficacy could be induced by Mycobacterium tuberculosis H37Ra in mice, while couldn't transcend BCG.
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