活细胞成像技术实时追踪荧光蛋白标记的分枝杆菌的胞内增殖  

Real-time tracking of intracellular mycobacteria proliferation by fluorescent protein marker and live cell imaging

作　　者：乔可[1] 王晴岚[1] 李紫煜 牛辰[1] 

机构地区：[1]复旦大学基础医学院教育部/卫生部医学分子病毒学重点实验室,上海200032 [2]北京东胜创新生物科技有限公司分子影像部,北京100085

出　　处：《复旦学报（医学版）》2014年第3期384-388,共5页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金青年项目(81201256);supported by the Youth Program of National Natural Science Foundation of China(81201256)

摘　　要：目的 利用绿色荧光蛋白（green fluorescent protein,GFP）标记和活细胞显微成像的方法,实时追踪分枝杆菌在巨噬细胞内的增殖过程。方法 用经GFP标记的野生型海分枝杆菌（Mycobacterium marinum,Mm）分别侵染未激活和γ-干扰素激活的巨噬细胞,使用活细胞显微成像系统连续记录胞内分枝杆菌的增殖动态,同时优化不同细胞接种浓度和感染复数（multiplicity of infection,MOI）对胞内菌增殖动态的追踪效果。结果 使用GFP标记和活细胞成像的方法可以实时追踪胞内分枝杆菌的增殖过程和形态学变化。γ干扰素对Mm在巨噬细胞中的增殖有抑制作用。当接种巨噬细胞浓度为1×105/孔,MOI为1时,可直观清晰地分析实时追踪图像中胞内菌的增殖过程。结论 通过活细胞成像技术分析荧光蛋白标记的分枝杆菌,可实时追踪巨噬细胞内分枝杆菌增殖过程和形态学变化,是一种体外分析胞内分枝杆菌增殖的理想方法,可应用于其他相关病原菌和宿主相互作用及影响因素的研究。Objective To track real-time proliferation of intracellular mycobacteria using green fluorescent protein （GFP） marker and live cell imaging.Methods Inactivated and interferon-γ activated macrophages were infected by Mycobacterium marinum （Mm） with GFP marker.Then,intracellular mycobacteria proliferation was continuously recorded by live cell microscopic imaging system.Different concentration of cells were inoculated and infected with Mm by series of multiplicity of infection （MOI） to identify the optimized experimental condition to track intracellular mycobacteria proliferation.Results By combining GFP marker and live cell imaging,the intracellular proliferation and morphological changes of mycobacteria can be tracked in real-time.Interferon-γ showed inhibitory effects on the proliferation of mycobacterium in macrophages.When the inoculation concentration of macrophages was 1×105/well,MOI was 1,the acquired images are most suitable for analyzing.Conclusions Live cell imaging is a powerful tool for investigating intracellular mycobacteria proliferation and morphological changes with GFP marker.It can be applied to other related studies,including the interaction of pathogen and host.

关 键 词：海分枝杆菌(Mm) 巨噬细胞 活细胞成像 细菌增殖 感染复数(MOI) 

分 类 号：R378.91[医药卫生—病原生物学]