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目的:研究阴道巨噬细胞清除后小鼠对布鲁氏菌疫苗的免疫应答机制。方法:BALB/c小鼠60只随机分为3组,每组20只,实验组小鼠阴道内滴注脂质体包裹的clodronate以清除阴道巨噬细胞,3d后阴道滴注10μL布鲁氏菌疫苗(含6×108个布鲁氏菌)。同时设空脂质体对照组(不含clo-dronate)和PBS对照组。依次在12、24、36、72h收集小鼠阴道、髂内淋巴结和血液。采用免疫组织化学染色法观察阴道巨噬细胞清除效果和树突状细胞(DC)的迁移,并用ELISA法检测血清中IFN-γ和IL-4的含量。收集小鼠腹腔液制备巨噬细胞进行细胞培养,1.2×106个巨噬细胞中加入布鲁氏菌疫苗10μL,于不同时间点对巨噬细胞进行姬姆萨染色,观察巨噬细胞的变化。结果:清除巨噬细胞组小鼠接种布鲁氏菌疫苗后,髂内淋巴结中S-100阳性DC数量没有明显的变化,IFN-γ和IL-4的含量也没有明显的升高。非清除组中,不同的时间点,髂内淋巴结中S-100阳性DC明显增多,与PBS对照组相比,IFN-γ含量显著增高,而IL-4则没有明显变化。体外巨噬细胞负载布鲁氏菌疫苗发现,随着时间的延长,巨噬细胞吞噬布鲁氏菌逐渐增多,在12h,巨噬细胞开始出现坏死性凋亡。结论:小鼠在对布鲁氏菌疫苗的免疫应答中,DC的迁移是巨噬细胞依赖性的,并且这种依赖性有可能是由布鲁氏菌抗原成分在细胞间间接传递引起的。
OBJECTIVE: To study the immune response to Brucella vaccine in mice after vaginal macrophage clearance. Methods: Sixty BALB / c mice were randomly divided into three groups with 20 mice in each group. The mice in the experimental group were intraperitoneally instilled with liposome-encapsulated clodronate to remove vaginal macrophages. After 3 days, vaginal instillation of 10 μL Brucella Bacterial vaccine (containing 6 × 108 Brucella). At the same time set empty liposome control group (without clo-dronate) and PBS control group. Mouse vagina, iliac lymph nodes and blood were collected at 12, 24, 36, 72 h. Immunohistochemical staining was used to observe the clearance of macrophages and the migration of dendritic cells (DCs). The contents of IFN-γ and IL-4 in serum were detected by ELISA. Mice peritoneal fluid were collected to prepare macrophages for cell culture. Brucella vaccine (10 μL) was added to 1.2 × 106 macrophages, and macrophages were harvested at different time points to observe the changes of macrophages. Results: The number of S-100-positive DCs in the internal iliac lymph nodes did not change significantly and the contents of IFN-γ and IL-4 did not increase significantly after macrophage-depleted mice were vaccinated with brucella. At different time points, the number of S-100 positive DCs in the iliac lymph nodes increased significantly in the non-ablation group compared with the PBS control group, whereas the level of IFN-γ was significantly increased in the non-ablation group, but not in the IL-4 group. In vitro macrophage-loaded Brucella vaccines, Brucella phagocytosis of macrophages gradually increased with time, macrophages began to appear necrotic apoptosis at 12h. CONCLUSIONS: In mice immunized with Brucella vaccine, DC migration is macrophage-dependent and this dependence may be caused by indirect transmission of Brucella antigen components between the cells.