肺炎克雷伯菌肝脓肿小鼠模型的制备与评价

Construction and evaluation of Klebsiella pneumoniae liver abscess mouse model

目的构建稳定且可靠的肺炎克雷伯菌肝脓肿(KPLA)小鼠模型,为该疾病的后续研究提供依据。方法选择肺炎克雷伯菌(KP)临床分离菌株181608为实验菌株,将不同浓度的细菌悬液通过腹腔注射,利用改良寇式法计算半数致死量(LD50),以1/2的LD50浓度注射C57BL/6J小鼠。通过小鼠一般情况、体质量变化、白细胞计数、HE染色以及肝脏菌落计数等指标综合评价该模型。结果根据改良寇式法计算出KP在C57BL/6J小鼠中的LD50约为104 CFU/ml。小鼠感染细菌24 h取肝脏脓肿部位的细菌培养结果为阳性,通过菌落形态、拉丝试验阳性和质谱结果鉴定致病菌为KP。HE染色结果提示小鼠感染后肝脏出现大面积坏死以及炎性细胞的大量浸润。随着感染时间的延长,小鼠精神状况变差,加之进食量的减少,体质量逐渐降低。检测造模小鼠的白细胞数高于对照组,差异有统计学意义(P<0.05)。除此之外,谷丙转氨酶(ALT)、谷草转氨酶(AST)和炎症因子[白细胞介素(IL)-1β、IL-5和肿瘤坏死因子(TNF)-α]在肝脓肿组明显升高,且RT-qPCR结果也显示炎性和趋化因子[IL-1β、IL-6、IL-10和单核细胞趋化蛋白(MCP)-1]的mRNA水平高于对照组,差异有统计学意义(P<0.05)。结论通过腹腔注射成功建立KPLA小鼠感染模型,为肝脓肿的研究提供稳定和易复制的动物模型。

Objective To establish a stable and reliable mouse model of Klebsiella pneumoniae liver abscess(KPLA)for deep research.Methods The clinical isolates of Klebsiella pneumoniae(KP)181608 were selected as the experimental strains.Mice were intraperitoneally injected with bacterial suspensions of different concentrations.Lethal dose 50(LD50)was calculated by the modified coon method,and the concentration of liver abscess model mice was 1/2 LD50.The general condition,weight change,blood routine examination,HE staining and liver bacterial burden were recorded to comprehensively evaluate the model and verify its effect.Results The LD50 of KP was 104 CFU/ml according to the improved Karber′s method.The result of bacterial culture from the site of liver abscess was positive,and the infected bacteria was identified as KP by colony morphology,positive wire-drawing test and mass spectrometry.The results of HE staining suggested large area of necrosis and infiltration of inflammatory cells in liver of KLA mice.With the extension of the infection time,the mental state of KLA mice became worse,coupling with the decrease of food intake,eventually the weight obviously decreased.The number of leukocytes in infected mice were higher than that in the control group,and the difference was statistically significant(P<0.05).In addition,alanine aminotransferase(ALT),aspartate aminotransferase(AST)and inflammatory factors[interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)]significantly increased in KLA group,and RT-qPCR results also showed higher mRNA levels of inflammatory and chemokine factors[IL-1β,IL-6,IL-10 and monocyte chemotactic protein-1(MCP-1)]than those of the control group,and the difference was statistically significant(P<0.05).Conclusion The KPLA mouse is successfully established by intraperitoneal injection,providing a stable and easy-to-replicate animal model of liver abscess for further study.