粪肠球菌体外根尖生物膜模型的建立及形态学研究

摘要

Objective This study aims to develop and observe a model system of the periapical biofilm structure of Entero-coccus faecalis (E. faecalis). Methods A total of 24 intact human single-rooted premolars extracted for orthodontic reasons were collected and randomly divided into eight groups (n=3). The specimens were subjected to ultraviolet disinfection, ino-culated with E. faecalis (ATCC 29212) suspension adjusted to 1×108 CFU·mL-1, and incubated at 37 ℃ for 1, 2, and 7 d. Specimen groups were prepared for scanning electron microscope to examine the biofilm formation. The specimens in the confocal laser scanning microscope (CLSM) groups were stained with propidium iodide (PI) and ConA-fluorescein iso-thiocyanate (ConA-FITC) to examine the biofilm formation. The images were randomized, and biofilm coverage (%) was assessed using Photoshop CS5. Results The biofilm coverage (%) on the cementum increased with increasing incubation period. The biofilm coverage of the 7 d group was significantly higher than those of the 1 and 2 d groups (P<0.05). The values of the latter two groups were not significantly different (P>0.05). Dense aggregations composed of E. faecalis and the amor-phous matrix were observed on the root cementum surfaces of the specimens in the 7 d group. The bacteria were stained red by PI, and the matrix was stained green by ConA-FITC under CLSM observation. The biofilm coverage (%) on the samples in the 7 d group was 17.23%±1.52%, showing multi-level space structure and water channels. Conclusion E. faecalis forms bacterial biofilms on the root cementum surface in 7 d. The biofilms were composed of E. faecalis and the amorphous matrix.%目的： 通过体外实验建立根尖生物膜模型，观察粪肠球菌根尖生物膜模型的形态及结构。方法 选取人新鲜拔除的单根离体牙24颗，随机分为8组，每组3颗，浸泡于粪肠球菌ATCC 29212菌悬液中，分别于1、2、7 d建立根尖生物膜模型。使用扫描电子显微镜（SEM）观察生物膜形成过程；使用碘化丙啶（PI）和刀豆球蛋白A异硫氰酸荧光素（ConA-FITC）对生物膜细菌及细胞外基质进行双染色，通过激光共聚焦扫描电子显微镜（CLSM）观察其形态和结构；计算生物膜覆盖率。结果 随时间的增加，根尖样本表面细菌覆盖面积增加，7 d组与1、2 d组间的差异存在统计学意义（P<0.05），1 d与2 d组间的差异无统计学意义（P>0.05）。7 d组形成生物膜结构，其样本表面的覆盖率为17.23%±1.52%，样本表面有大量细菌附着，细菌被细胞外基质包绕，并呈群落分布。生物膜中的细菌被PI染成红色，细胞外基质被ConA-FITC染成绿色，可见多层次的空间结构以及亲水性通道。结论 粪肠球菌根尖生物膜于培养7 d时形成完整的生物膜结构，由细菌群落及其周围大量的不定型基质组成。