白色念珠菌生物膜体外模型的建立及不同检测方法比较

目的建立白色念珠菌生物膜体外模型,为进一步研究白色念珠菌生物膜的耐药机制及干预提供基础。方法平板法培养白色念珠菌生物膜,利用扫描电镜、激光共聚焦显微镜和荧光显微镜等多种检测方法观察生物膜形成情况。结果白色念珠菌能够在玻片上形成典型的生物膜,通过荧光显微镜、扫描电镜和激光共聚焦显微镜下观察白色念珠菌随着时间增加逐渐增加,48 h形成初步生物膜,72 h结构更加复杂和成熟。ISA软件定量化分析显示,SYTO9/PI荧光探针标记的生物膜模型的区域孔径(AP)在24、48和72 h分别为0.95±0.06、0.89±0.01和0.83±0.01,平均扩散距离(ADD)分别为1.16±0.13、1.26±0.06和2.43±0.76,结构熵(TE)分别为4.87±0.34、5.18±0.35和5.47±0.16,两两比较,差异均有统计学意义(P0.05)。Im age-Pro P lus 6.0软件分析显示,生物膜内真菌死亡率分别为(34.71±2.72)%、(36.63±4.20)%和(47.41±2.53)%,与24 h及48 h相比,72 h真菌死亡率增加差异具有统计学意义(P0.05)。结论平板法能够较好地建立白色念珠菌生物膜体外模型,并可利用多种方式检测。

Establishment of the model of Candida albicans biofilm in vitro and the comparison of different detections

Objective To establish Candida albicans biofilm in vitro,build foundation for further study on the resistance mechanism and treatments.Method Plate culture method was used to establish Candida albicans biofilm which was identified by scanning electron microscope（SEM） and confocal laser scanning microscope（CLSM）.Result The model of Candida albicans biofilm in vitro established on coverslip.Observation showed that the biofilm formation was observed about 48 h after culture,and its architecture became more mature and complex after 72 h.The quantitative data from ISA software showed that in the biofilm models stained with SYTO9/PI at different time intervals（24 h,48 h,72 h）,the areal porosity（AP） were 0.95±0.06,0.89±0.01 and 0.83±0.01,respectively;the average diffusion distance（ADD） were 1.16±0.13,1.26±0.06 and 2.43±0.76,respectively;the textural entropy（TE） were 4.87±0.34,5.18±0.35 and 5.47±0.16,respectively.There were significant differences between any two groups（P〈0.05）.The quantitative data from Image-Pro Plus 6.0 software showed that the mortality of Candida albicans were（34.71±2.72）%,（36.63±4.20）% and（47.41±2.53）%,respectively.There were significant differences between the 72 h group and the other two groups（P〈0.05）.Conclusion Candida albicans has biofilm-forming ability,the biofilm can be identified and analyzed by many methods.