小鼠鲍曼不动杆菌肺感染模型的制备

Establishment of Acinetobacter baumannii-induced pneumonia model in mice

目的构建小鼠鲍曼不动杆菌(A.baumannii)诱导的肺炎模型,研究A.baumannii致病的分子机制。方法实验分为正常对照组、环磷酰胺预处理组、正常小鼠接种组和免疫缺陷小鼠接种组。利用免疫抑制剂环磷酰胺预处理实验雄性C57BL/6小鼠制备免疫缺陷小鼠模型,使用临床重症监护病房(ICU)分离的A.baumannii制备新鲜菌液(1×108集落形成单位/m L),通过气道接种方法接种至小鼠,分别于接种后6、24、72 h,进行小鼠肺及肺泡灌洗液白细胞和中性粒细胞计数,HE染色观察肺组织炎症变化,ELISA检测试剂盒检测血清粒细胞巨噬细胞集落刺激因子(GM-CSF)、γ干扰素(IFN-γ)、白细胞介素1β(IL-1β)、IL-2、IL-4、IL-5、IL-6、IL-10、IL-12、肿瘤坏死因子α(TNF-α)水平。结果免疫功能正常的小鼠感染A.baumannii72 h后,肺内细菌基本被清除,而免疫抑制剂处理的小鼠接种A.baumannii后,肺及血液中细菌计数持续增加;正常小鼠感染6 h后,体内白细胞计数和中性粒细胞升高,至72 h下降,而经过免疫抑制剂处理的小鼠感染后,肺及血液中白细胞计数和中性粒细胞持续升高;感染72 h后,免疫功能正常小鼠肺部有轻微炎症,免疫缺陷小鼠肺部炎症较为明显;正常小鼠感染6 h后,血清中上述细胞因子含量均增加,至72 h下降,而经过免疫抑制剂处理的小鼠感染后,肺及血液中上述细胞因子水平均持续升高。结论成功制备了A.baumannii诱导的小鼠肺部感染模型。

Objective To establish Acinetobacter baumannii（A. baumannii）-induced pneumonia models in C57 BL/6 mice,and study the molecule mechanism of A. baumannii infection. Methods Eighty C57 BL/6 mice were divided into normal control group,cyclophosphamide-treated group,A. baumannii infection group,and cyclophosphamide-pretreated A. baumannii infection group. Immunodeficient mice were prepared by injecting cyclophosphamide intraperitoneally.A. baumannii was isolated from intensive care unit（ ICU） and fresh bacteria（1 × 108 CFU/m L） were prepared. Each normal or immunodeficient mouse was inoculated with 50 μL A. baumannii through trachea. The lung,bronchoalveolar lavage fluid（BALF） and blood were collected at 6,24 and 72 hours after inoculation. The numbers of white blood cells（WBCs） and neutrophils were detected by cell counting. The histopathology of the lung was evaluated by HE staining. Cytokines such as granulocyte macrophage colony-stimulating factor（ GM-CSF）,interferon γ（ IFN-γ）,interleukin 1β（ IL-1β）,IL-2,IL-4,IL-5,IL-6,IL-10,IL-12,tumor necrosis factor α（ TNF-α） were detected by ELISA. Results A. baumannii was eliminated within72 hours after infection in normal mice,whereas the bacteria continued to replicate rapidly in the lungs and blood in the immunodeficient mice. The numbers of WBCs and neutrophils were elevated markedly 6 hours post infection,and return to the normal within 72 hours. However,the numbers of WBCs and neutrophils continuously increased in cyclophosphamidepretreated A. baumannii infection group,and the pulmonary inflammatory was more severe than that in the normal mice. The cytokines of blood increased markedly 6 hours post infection,and then decreased until 72 hours. However,the cytokines continuously increased in cyclophosphamide-pretreated A. baumannii infection group. Conclusion A. baumannii-induced pneumonia models in C57 BL/6 mice were established successfully.