羊布氏菌强毒株16M感染小鼠骨髓树突状细胞模型的建立

Establishment of Bone Marrow-derived Dendritic Cell Model of Mice Infected with Virulent Brucella melitensi 16M Strain

目的建立羊布氏菌强毒株16M感染小鼠骨髓树突状细胞(Dendritic cell,DC)的模型。方法体外分离C57小鼠骨髓原代细胞,经粒细胞-巨噬细胞集落刺激因子(Granulocyte monocyte colony stimulating factor,GM-CSF)和重组人白细胞介素-4(Recombinant human interleukin-4,rhIL-4)诱导分化后,倒置显微镜观察DC形态,流式细胞术鉴定其分化程度。在体外建立羊布氏菌16M感染小鼠骨髓DC模型,间接免疫荧光试验和透射电镜进行鉴定,并进行感染后羊布氏菌16M的重培养及染色观察。结果培养第5天,细胞形态符合髓源DC,伸出长的树突样和伪足样突起;第7天,细胞呈半悬浮,周围有大量突起;体外培养DC的纯度约达70%,符合试验要求。DC在感染后12 h具有最强的吞噬能力,胞内细菌量最少。重培养后经染色,可观察到羊布氏菌16M典型的细菌形态。结论成功构建了羊布氏菌感染小鼠骨髓DC模型,为进一步研究布氏菌与DC的相互作用及胞内寄生机制奠定了基础。

Objective To establish a bone marrow-derived dendritic cell（DC） model of mice infected with virulent Brucella melitensi 16M strain.Methods Primary bone marrow cells of C57 mice were isolated in vitro,of which the differentiation was induced by granulocyte monocyte colony stimulating factor（GM-CSF） and recombinant human interleukin-4（rhIL-4）,then observed for morphology of DCs under invert microscope,and determined for differentiation level by flow cytometry.The DC model of mice infected with virulent B.melitensi 16M strain was established in vitro,identified by IFA and transmission electron microscopy,and subjected to re-culture and staining of B.melitensi 16M strain after infection.Results On the 5th day after culture,the morphology of cells was consistent with that of bone marrow-derived DCs,with long dendritic and pseudopod-like enations.On the 7th day,the cells were semi-suspended,with a large quantity of enations all around.The purity of DCs cultured in vitro reached about 70%,which met the requirements for test.DCs showed the strongest phagocytosis and the smallest amount of intracellular bacteria 12 h after infection.Typically bacterial morphology of B.melitensi 16M strain was observed by staining after re-culture.Conclusion The bone marrow-derived DC model of mice infected with virulent B.melitensi 16M strain was successfully constructed,which laid a foundation of further study on interaction of B.melitensi and DCs as well as the mechanism of intracellular parasitism.