激光共聚焦显微镜观察维拉帕米对HMME—PDT抑制混合菌菌斑生物膜作用的影响

Study of the effective of effiux pump inhibitors in photodynamic therapy inhibition the cariogenic bacteria in dental plaque biofilms

目的探讨细菌外排泵抑制剂（EPI）——维拉帕米对以血卟啉单甲醚（HMME）为光敏剂的光动力疗法（PDT）抵抗牙菌斑生物膜内主要致龋菌作用的影响。方法以变形链球菌、血链球菌、嗜酸性乳杆菌和黏性放线菌为实验菌株，建立牙菌斑生物膜模型。以维拉帕米作为细菌外排泵抑制剂，根据在PDT过程中加入维拉帕米和光敏剂的先后顺序，实验分为5组：A组为同时加入光敏剂＋维拉帕米组，B组为维拉帕米（先）＋光敏剂组（后）组，C组为光敏剂（先）＋维拉帕米（后）组，D组为单纯PDT处理组，E组为生理盐水处理组。激光照射后以激光共聚焦显微镜观察PDT作用后生物膜的变化。结果生理盐水处理组：生物膜内活菌数量占据大多数。以此组为正常细菌活力组作对比，单纯PDT处理后，红色荧光显著增加，细菌失去了积聚能力，多呈散乱分散状态；与单纯PDT组相比，加入维拉帕米的PDT组绿色荧光染色增多，细菌活力增加，其中先加入维拉帕米后加入光敏剂组，绿色荧光染色明显增多，红色荧光减少，说明活菌相对活力较高。结论实验表明，HMME。PDT在对混合菌菌斑生物膜中的细菌有抑制效应，细菌外排泵抑制剂对HMME—PDT抑制牙菌斑生物膜内主要致龋菌有抑制作用；且前期维拉帕米给药能够显著抑制PDT治疗龋病的效果．

Objective To Study the effect of efflux pump-inhibitors（EPI）-Verapamil in photodynamic therapy （PDT） using hematoporphyrin monomethylether （HMME） as photosensitizer on the cariogenic bacteria in dental plaque biofilms. Methods According to the administrator order of the verapamil and photosensitizer in PDT, streptococcus mutans, streptococcus sanguis, eosinophilic lactobacillus and actinomyces viscosus were used to establish the dental plaque biofllm model. The experiment was divided into five groups, group A was incubated with the photosensitizer and verapamil group, group B using verapamil before incubated the photosensitize, group C suing photosensitizer before incubated verapamil, group D with PDT only, group E was control group. After laser treatment, the influence of the dental plaque biofilms was observed by confocal laser scanning microscope. Results As saline-treated group is a group of normal the dental plaque biofilms. In PDT only group, compared with the saline group, red fluorescence increased significantly, the bacteria lose accumulation capacity, and were isolated and scattered in dispersed state. In PDT plus verapamil group, compared with only PDT group, green staining increased, bacterial activity increased. In group B, cells were incubated with verapamil before incubated the photosensitizer group, green staining increased significantly, red fluorescence reduced, indicating live bacteria increased, and bacteria activity was significantly increased. Conclusion PDT is an effective method in eliminating cariogenic bacteria of dental plaque biofilms. Bacterial efflux pump inhibitors can lower HMME-PDT inhibition cariogenic bacteria in dental plaque biofilm, and pre-verapamil administration could significantly inhibit the effect of PDT treatment of dental caries.