细胞膜微结构域中胆固醇对小鼠巨噬细胞摄取土拉-微生物与感染.pdfVIP

微生物 与感染 2009 年 12 月第 4 卷第 4 期 Journal of Microbes and Infection, December 2009, Vol. 4, No. 4 ·223 · 细胞膜微结构域中胆固醇对小鼠巨噬细胞摄取土拉 弗朗西斯菌的作用 潘欣, 李广波, 瞿旻, 赵子夜, 李晗, 戚中田 第二军医大学微生物学教研室, 上海市医学生物防护重点实验室, 上海 200433 摘 要: 本文旨在探讨细胞膜微结构域中胆固醇在小 鼠巨噬细胞摄取土拉弗朗西斯菌 LVS 株 中的作用。用 电穿孔法将穿梭质粒 pFNLTP6 gro-gfp 导入土拉弗朗西斯菌LVS株; 细胞胆固醇用菲律平Ⅲ染色, 结合单克隆 抗体的小窝蛋白-1 用键合了Alexa 594 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 株感染; 用甲基- β-环糊精干扰富含脂质的胞膜, 损耗胆固醇, 以评估膜微结构域的损伤对土拉弗朗西 斯菌入侵的影响, 菲律平Ⅲ用于检测胆固醇损耗的效果。结果显示, 细胞胆固醇在早期与摄取的土拉弗朗西 斯菌疫苗株共定位, 膜微结构域相关成分小窝蛋白- 1 与两者接触密切; 土拉弗朗西斯菌入侵巨噬细胞需要胆 固醇丰富的膜结构域。结果提示, 胆固醇丰富的膜微结构域和小窝蛋白- 1在土拉弗朗西斯菌早期进入巨噬 细胞过程中发挥重要作用。 关键词: 巨噬细胞; 土拉弗朗西斯菌; 膜微结构域; 小窝蛋白- 1; 胆固醇 Role of cholesterol of membrane microdomain in the uptake of Francisellatularensis by mouse macrophages PAN Xin, LI Guang-Bo, QU Min, ZHAO Zi-Ye, LI Han, QI Zhong-Tian Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai 200433, China Abstract: The purpose of the current study was to evaluate the role of cholesterol of the membrane microdomain in the uptake of Francisella tularensis ( F. tularensis) LVS by mouse macrophages. A shuttle plasmid, pFNLTP6 gro-gfp, was transformed by electroporation to F. tularensis LVS. Cell cholesterol was stained with Filipin Ⅲ, and caveolin- 1 was detected with monoclonal antibody and visualized with Alexa 594 conjugated goat anti-mouse antibodies. F. tularensis LVS infection was analyzed using a fluorescence microscope equipped with a motorized Z-focus. In order to evaluate the effect of depletion of the membrane microdomain on F. tularensis entry into macrophages, interference with lipid-rich plasma membrane through the depletion of cholesterol was performed by methyl- β-cyclodextrin. The cholesterol-binding agent, Filipin III, was used to detect the effect of cholesterol depletion. The results showed that cell cholesterol was co-localized with F. tularensis live vaccine strain