绿色荧光蛋白标记铜绿假单胞菌生物膜形成动态观察及结构定量分.docVIP

绿色荧光蛋白标记铜绿假单胞菌生物膜形成动态观察及结构定量分 　 作者：陈波曼 余加林 刘官信 胡琳燕 李芳 杨华 【摘要】 目的 探讨铜绿假单胞菌细菌生物膜(biofilm，BF)形成发展过程中空间立体结构变化，以及BF形成过程中与其生物学行为之间的相互联系。方法 体外建立6h和1、3及6d等4个时间组铜绿假单胞菌PAO1菌株BF模型，通过绿色荧光蛋白(green fluorescence protein，GFP)标记，结合激光共聚焦显微镜(confocal laser scanning microscopy，CLSM)摄取BF形成发展各阶段不同层面的图片堆，经图像结构分析软件(image structure analyer，ISA)分析获得PAO1菌株BF相关空间结构参数定量化数据。结果 ①CLSM结合GFP标记，实现了对PAO1菌株BF动态形成过程的观察；②ISA软件定量化分析显示，随着BF发展，厚度不断增加，前3d增加程度(19.6μm)明显大于后3d(6.1μm)，区域孔率(areal porosity，AP)、平均扩散距离(average diffusion distance，ADD)和结构熵(textural entropy，TE)在BF形成的6h和6d分别为：0.98±0.01和0.92±0.02，呈现下降趋势；1.00±0.009和1.06±0.027，虽变化幅度不大，但亦呈现逐渐增加趋势；0.7±0.08和4.3±0.09，呈明显上升趋势。结论 ISA程序可以表征PAO1菌株BF的空间结构特征，对细菌BF结构的定量化分析也有助于探索BF形成过程与其生物学行为之间的相互联系。 【关键词】 生物膜； 铜绿假单胞菌； 定量分析； 结构 ABSTRACT Objective To quantify the sequential development of biofilm spatial structure in biofilm process with Image Structure Analyer (ISA) software, which will provide assistant to further research on the biological behavior of biofilm. Methods P.aeruginosa PAO1 biofilm model in vitro was constructed on glass slice and biofilm development was monitored in different time intervals (6 hours, 1 day, 3 days and 6 days). The fluorescence images stack of different layer in biofilm model were obtained by confocal laser scanning microscopy (CLSM), based on fluorophores from PAO1 with green fluorescent protein (GFP) genetically tagged. Quantitative parameters describing biofilm spatial structure could be acquired after the image information was calculated by ISA software. Results ①The PAO1 biofilm process was investigated successfully by CLSM after genetically tagged with GFP. ②The quantitative data from ISA software showed that the thickness of biofilm accumulated during biofilm growth, the rate was more obvious during the first 3 days than the latter 3 days (19.6μm vs. 6.1μm); meanwhile the areal porosity (AP) decreased from 0.98±0.01 at 6h to 0.92±0.02 at 6d, the average diffusion distance (ADD) increased slightly from 1.00±0.009 at 6h to 1.06±0.