氨基酮戊酸孵育不同时间对光动力疗法抑制痤疮丙酸杆菌生物膜效应的影响

Effect of different incubation time of aminolevulinic acid on photodynamic inhibition of Propionibacterium acnes biofilms

目的探讨氨基酮戊酸(ALA)孵育不同时间对ALA光动力疗法(ALA-PDT)抑制痤疮丙酸杆菌生物膜效应的影响。方法在预先放置细胞爬片的24孔和96孔细胞培养板中构建痤疮丙酸杆菌生物膜,使用共聚焦激光扫描显微镜(CLSM)观察生物膜的形成情况,用甲基四氮盐(XTT)法观察生物膜生长活力情况。确定生物膜体外模型构建成功后,分成6组,阴性对照组不加入ALA、不照光;ALA组仅予ALA孵育30 min,不照光;LED组仅予照光但不加入ALA;ALA-PDT1组、ALA-PDT2组、ALA-PDT3组分别与ALA孵育15、30、60 min后,接受LED光照射。处理完成后采用CLSM检测生物膜结构、死菌/活菌比例,XTT法观察生物膜生长活力差异。组间差异比较采用单因素方差分析及LSD-t检验。结果CLSM观察显示,痤疮丙酸杆菌生物膜体外模型构建成功。ALA-PDT1组、ALA-PDT2组、ALA-PDT3组死菌/活菌比值分别为0.90±0.16、1.75±0.19和2.57±0.32,均显著高于阴性对照组(0.31±0.01,t值分别为55.56、138.62、74.64,均P<0.001);生物膜活力值分别为0.35±0.02、0.26±0.02和0.18±0.01,均显著低于阴性对照组(0.43±0.00;t值分别为35.66、2.64、110.96,均P<0.001)。CLSM显示,痤疮丙酸杆菌生物膜在ALA-PDT作用下结构被破坏,且随着ALA孵育时间延长,生物膜破坏越严重。结论ALA孵育时间延长会增强ALA-PDT抑制痤疮丙酸杆菌生物膜效应。

Objective To investigate the effect of different incubation time of aminolevulinic acid(ALA)on photodynamic inhibition of Propionibacterium acnes biofilms.Methods Propionibacterium acnes biofilms were formed in 24-well plates with pre-placed cell slides and 96-well plates.The formation of the biofilm structure was observed by confocal laser scanning microscopy(CLSM),and the growth activity of the biofilm was assessed by the tetrazolium salt XTT assay.The in vitro successfully constructed biofilm models were divided into 6 groups:negative control group receiving neither ALA treatment nor LED radiation,ALA group incubated with ALA alone for 30 minutes,LED group receiving LED radiation alone,ALA-PDT1 group,ALA-PDT2 group and ALA-PDT3 group incubated with ALA for 15,30 and 60 minutes respectively followed by LED radiation.After the treatment,CLSM was performed to observe the biofilm structure,as well as to determine the dead/living bacteria ratio,and XTT assay to assess the growth activity of the biofilm.Differences among groups were analyzed using one-way analysis of variance and least significant difference-t test.Results CLSM showed that the Propionibacterium acnes biofilm model was successfully constructed in vitro.The dead/living bacteria ratios were 0.90±0.16,1.75±0.19,and 2.57±0.32 in the ALA-PDT1 group,ALA-PDT2 group and ALA-PDT3 group respectively,which were significantly higher than the dead/living bacteria ratio in the negative control group(0.31±0.01;t=55.56,138.62,74.64,respectively,all P<0.001);the biofilm viability value was significantly lower in the ALA-PDT1 group,ALA-PDT2 group and ALA-PDT3 group(0.35±0.02,0.26±0.02,0.18±0.01,respectively)than in the negative control group(0.43±0.00;t=35.66,2.64,110.96,respectively,all P<0.001).CLSM showed that the structure of the Propionibacterium acnes biofilm was destroyed under the action of ALA-PDT,and the destruction was aggravated with the prolongation of incubation time of ALA.Conclusion The prolongation of incubation time of ALA can enhance the inhibitory effect of ALA-PDT on Propionibacterium acnes biofilms.