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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 effect of Francisella tulara LVS on the plasma membrane microarray in murine macrophages. Methods The shuttle plasmid pFNLTP6 gro-gfp was introduced into Francisella tularensis LVS by electroporation. Cell membranes were stained with FM4-64 and monoclonal antibody-conjugated mouse monoclonal protein-1 was developed with goat anti-mouse secondary antibody conjugated with Alexa594 fluorescein. Analysis of Francisella tularensis LVS infection with a z-axis electrofocusing fluorescence microscope. Interference of lipid-rich plasma membrane depletion of cholesterol with methyl-β-cyclodextrin to assess the effect of plasma membrane damage on the invasion of Francisella. Filipin III is used to detect the effect of cholesterol depletion. As a result, the F. tularensis strain could induce the pseudopodia in the membrane domain to absorb bacteria into macrophages. Plasma membrane related microencapsulated protein-1 and Francisella-containing vesicles enter the host. Francisella invasion of macrophages requires a cholesterol-rich membrane domain. Conclusion Cholesterol-rich plasma membrane microdomains and microcystin-1 play an important role during the early phase of Francisella into macrophages.