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目的建立类鼻疽伯克霍尔德氏菌感染人肺癌上皮细胞A549的细胞模型。方法优化类鼻疽伯克霍尔德杆菌感染A549细胞的感染条件(如MOI、感染时间),通过活细胞工作站动态观察、Giemsa染色、激光共聚焦、透射电镜确证胞内感染和宿主细胞的形态变化,通过炎症因子IL-8和TNF-α的检测,分析病原菌入胞率和宿主反应性,评价该模型中病原菌侵入A549导致多核巨细胞(multinuclear giant cell,MNGC)形成的特点和病理损伤规律。结果透射电镜结果显示类鼻疽伯克霍尔德氏菌侵入到胞内的时间最早是4 h。通过Giemsa染色形态观察,类鼻疽伯克霍尔德氏菌感染后最早8 h可观察到典型MNGC的形成。随着感染时间的延长,MNGC形成率和炎症因子IL-8逐渐升高,而TNF-α在此模型中变化不明显。结论成功构建类鼻疽伯克霍尔德氏菌感染A549细胞模型。
Objective To establish a cell model of Burkholderia pseudomallei infection of lung cancer A549 cells. Methods The infection conditions (such as MOI, infection time) of Burkholderia pseudomallei-infected A549 cells were optimized. The intracellular infection and the morphological changes of the host cells were confirmed by dynamic observation with live cell workstation, Giemsa staining, confocal laser scanning microscopy and transmission electron microscopy. Through the detection of inflammatory cytokines IL-8 and TNF-α, the incidence of enteric pathogens and host reactivity were analyzed, and the invasion of A549 by this pathogen was evaluated to induce the formation of multinuclear giant cell (MNGC) and the pathological damage. Results Transmission electron microscopy showed that Burkholderia pseudomallei invaded intracellularly the earliest time was 4 h. The typical MNGC formation was observed at the earliest 8 h after Burkholderia pseudomallei infection by Giemsa staining. With the prolonging of infection time, the formation rate of MNGC and inflammatory cytokines IL-8 gradually increased, but the changes of TNF-αin this model were not obvious. Conclusion The model of Burkholderia bacillus infection in A549 cells was constructed successfully.