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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 model of murine bone marrow dendritic cells (DC) infected with 16M mutants of Mycobacterium veneseum. Methods The primary bone marrow cells of C57 mice were isolated and cultured in vitro. Granulocyte monocyte colony stimulating factor (GM-CSF) and recombinant human interleukin-4 (rhIL-4) After induced differentiation, DC morphology was observed by inverted microscope, and the differentiation degree was identified by flow cytometry. The bone marrow DCs of 16M-infected mice were established in vitro. Indirect immunofluorescence assay and transmission electron microscopy were used to identify the inbred strains of 16M. Results On the fifth day after culture, the morphology of the cells was consistent with the dendritic cells derived from the medulla oblongata and extended into the dendritic and pseudopodia-like processes. On the seventh day, the cells were semi-suspended and surrounded by a large number of protrusions. The purity of DC cultured in vitro was about 70% Meet the test requirements. DC had the strongest phagocytosis at 12 h after infection, with the least intracellular bacteria. After heavy culture after staining, can be observed in the typical 16M lamblia bacteria form of bacteria. Conclusion The bone marrow DC model in mice infected with B. lamblia was successfully constructed, which laid the foundation for the further study on the interaction between Brucella and DC and the intracellular parasitic mechanism.