口腔生物膜动态研究模型的建立和应用评价

Establishment and evaluation of the in vitro dynamic biofilm model

目的:模拟口腔菌斑生态环境,建立能够对生物膜形成过程连续观察的动态研究模型。方法:自制发酵罐和多出口连续培养室。以变形链球菌、血链球菌、乳酸杆菌、内氏放线菌为实验菌株,将羟磷灰石片、玻片等置于连续培养室中,通过调节温度、氧分压、pH值、清除率、循环流速及代谢底物等参数建立模型。以25mM蔗糖和蒸馏水进行系统测试。观察系统中实验菌CFU计数、pH动态变化等各项反应及不同环境下所形成生物膜的微结构变化。所得数据采用SPSS10.0软件包进行统计学分析,选用单因素方差分析的Dunnet t双侧检验,在α=0.05的情况下比较蔗糖加入组与对照组之间的差异。结果:随着蔗糖的给予,连续培养室中pH表现出相似于天然口腔的致龋环境,蒸馏水组pH值始终维持在7.5左右。而蔗糖组pH值呈明显的周期性波动,最低在4.5以下,但4～5h后,其pH值仍能逐步恢复到6.5左右。观察到细菌计数、生物膜形态的时间依赖性。随着处理液的加入,2组生物膜的活菌计数均明显增加,约72h趋于稳定。对120h生物膜,蔗糖组变形链球菌、乳酸杆菌的细菌计数显著高于蒸馏水组(P<0.05);蔗糖组变形链球菌计数最高,而蒸馏水组血链球菌计数最高。荧光显微镜观察发现,蔗糖组大量的胞外多糖基质和细菌成团、成片分布;蒸馏水组虽然也存在细菌和基质增多的时间依赖性,但膜细菌基质团块仍清晰可见。结论:本研究建立的体外模型能稳定控制有关参数,各观察指标均具有良好的重复性。模型的建立,实现了生物膜形成过程的动态观察。

PURPOSE： To establish a dynamic model which could simulate the ecologic environment of oral cavity and study the cariogenicity of oral biofilm, continuously observe the formation of oral biofilm.METHODS： The fermental vessel and chemostat were designed according to the purpose of the study. Four strains of oral bacteria, namely Streptococcus mutans, Streptococcus sanguis, Lactobacillus rhamnosus and Actinomyces naeslundii, were inoculated in the fermenter. The hydroxyapatite （HA） discs and glass slides were put in the chemostat. The temperature, oxide pressure, pH, dilution rate, circulation rate and substrate of the chemostat were carefully modulated. Distilled water and 25mM sucrose solution were used to test the stability of the system. At the beginning of the model establishment, the anabiosis bacteria were inoculated in fermenter full of fresh artificial saliva with a dilution rate of 10%. After the growth of bacteria in the fermenter was stable, the four-organism bacterial consortium was inoculated in the chemotat for 24 hours together with fresh artificial saliva （v/v=1：9）. The organisms in planktonic phase were examined everyday. When the populations of consortium got stable （about 72 hours）, the test solution was pulsed in the chemostat every 12 hours for 5 days. The HA discs and glass slides were observed every 24 hours for five times. The data was analyzed using SPSS 10.0 software package.Dunnet t test was used to compare the difference between the two groups at the level of α=0.05. RESULTS： Thedynamic model was successfully established on oral biofilm study. With the pulse of sucrose solution, the environment in the chemostat simulated the cariogenic environment in nature, and all data namely the bacteria population, the biofilm microstructure changed with solution pulse. CONCLSUIONS： The model established is proved to be stable and the parameters in the model could be successfully controlled. The model fulfills the dynamic observation of formation of biofilm and caries lesion in vitro.