改良恒化器中牙周致病菌和致龋菌的激光共聚焦显微镜动态观察

Dynamic observation of periodontal pathogens and cariogenic bacteria in modified chemostat using confocal laser scanning microscopy

目的 模拟口腔条件下研究牙周致病菌和致龋菌的动态关系。方法 选用牙龈卟啉单胞菌、伴放线放线杆菌、具核梭杆菌、中间普氏菌、变形链球菌、血链球菌、黏性放线菌和嗜酸乳杆菌8种牙周致病菌和致龋菌。按分组接种于模拟口腔的改良恒化器中 ,连续培养 1、2 4、4 8和 96h ,活菌革兰荧光染色与激光共聚焦显微镜结合测量羟基磷灰石圆片表面生物膜厚度 ,连续断层扫描及三维重建。结果 随时间变化各组生物膜厚度均显著增加 (P <0 0 0 1) ;同一时间点血链球菌生物膜明显厚于伴放线放线杆菌 ,8种菌明显比血链球菌和伴放线放线杆菌形成的生物膜厚。三维重建显示 ,G-牙周致病菌主要分布于G + 致龋菌菌团中或膜表层。结论 人工菌斑生物膜中G + 致龋菌首先定植 ,G -牙周致病菌的量和比例随时间增加 ;

Objective To study dynamic relation between periodontal pathogens and cariogenic bacteria under analogous oral enviroment Methods Eight periodontopathic and cariogenic bacteria of Porphyromonas gingivalis (Pg), Actinobacillus actinomycetemcomitans (Aa), Fusobacterium nucleatum (Fn), Provotella intermedium (Pi), Streptococcus mutans (Sm), Streptococcus sanguis (Ss), Actinomyces viscosus (Av) and Lactobacillus acidophilus (La) were used These eight strains were cultured in modified chemostat under analogous oral environment which contained 600 ml modified BM medium supplemented with 2 5 g/L porcine gastric mucin, respectively After 1, 24, 48 and 96 h, optical sectioning of plaque biofilms on removable and replaceable hydroxyapatite disks was analyzed by the combination of live bacterial Gram fluorescence staining and confocal laser scanning microscopy Biofilm thickness and reconstruction of the three dimensional architecture of plaque biofilms were made Results Biofilm thickness increased significantly with time ( P <0 001) Biofilms of Aa were thinner than those of Ss and eight specie biofilms were thicker than those formed by Ss and Aa per time point Three dimensional images showed periodontal pathogens mainly occurred in cariogenic bacterial complex or on the biofilm surface Conclusions Gram positive cariogenic species initially predominated in artificial plaque, followed by the increasing proportions of Gram negative periodontal pathogens The relation between microecological balance among bacteria and diseases is worthy of further studies