质膜微区对土拉弗朗西斯菌LVS感染鼠巨噬细胞的影响(英文)  

Influence of membrane microdomin on infection process of mouse macrophages by Francisella tularensis LVS

作　　者：潘欣 祁建城 李广波 贾文凯 瞿旻 赵子夜 

机构地区：[1]Department of Microbiology, Second Military Medical University, Shanghai 200433, China [2]China National Center for Biological Protection Engineering, Tianjin 300161, China

出　　处：《解放军医学杂志》2009年第7期833-837,共5页Medical Journal of Chinese People's Liberation Army

基　　金：Supported by the Foundation for Supporting Returned Overseas Chinese Scholars,State Education Ministry (2008-890-2);the National Natural Science Foundation of China (30170891)

摘　　要：目的评估土拉弗朗西斯菌LVS入侵鼠巨噬细胞过程中质膜微区的作用。方法用电穿孔法将穿梭质粒pFNLTP6 gro-gfp导入土拉弗朗西斯菌LVS。细胞膜用FM4-64染色,结合单抗的微囊蛋白-1用键合了Alexa594荧光素的羊抗鼠二抗显色。用Z轴电动聚焦的荧光显微镜分析土拉弗朗西斯菌LVS感染。用甲基-β-环式糊精干扰富含脂质的胞膜损耗胆固醇,以评估质膜微区的损伤对弗朗西斯菌入侵的影响。菲律平Ⅲ用以检测胆固醇损耗的效果。结果土拉弗朗西斯菌疫苗株可以诱导膜结构域伸出伪足将细菌吸收进入巨噬细胞。质膜微区相关成分微囊蛋白-1与含有弗朗西斯菌的囊泡一同进入宿主。弗朗西斯菌入侵巨噬细胞需要胆固醇丰富的膜结构域。结论胆固醇丰富的质膜微区和微囊蛋白-1在弗朗西斯菌早期进入巨噬细胞期间发挥重要作用。Objective To evaluate the role of membrane microdomain on the entry process of Francisella tularensis LVS infecting mouse macrophages. Methods A shuttle plasmid pFNLTP6 gro-gfp was introduced to F. tularensis LVS by electroporation method. Membrane was stained with FM4-64, and caveolin-1 was detected with mono-antibody and visualized with Alexa Fluor 594 conjugated goat-ant-mouse antibodies. F. tularensis LVS infection was analyzed by a fluorescence microscope equipped with a motorized Z-focus. In order to evaluate the depletion of membrane microdomain induced by Francisella entering macrophages, interference with lipid-rich plasma membrane through the depletion of cholesterol was performed by treatment with methyl beta cyclodextrin. The cholesterol-binding agent Filipin Ⅲ was used to detect the effect of cholesterol depletion. Results It was shown that the live vaccine strain of F. tularensis induced membrane domains ruffling for successful entry into macrophages. Membrane microdomains-associated components such as caveolin- 1 were incorporated into Francisella containing vesicles when entering the host cell. Francisella requires cholesterol-rich membrane domains for entering into macrophages. Conclusion It is suggested that cholesterol-rich membrane microdomains and eavcolin-1 play an important role in the early stage of Francisella entering into macrophages.

关 键 词：巨噬细胞 土拉弗朗西斯菌 质膜微区 微囊蛋白-1 胆固醇 

分 类 号：R378.19[医药卫生—病原生物学]