变异链球菌生长及生物膜形成能力的初步研究

发布时间：2018-06-28 04:41

  本文选题：变异链球菌 + 报告株　； 参考：《第四军医大学》2010年硕士论文

【摘要】：龋病是常见的口腔感染性疾病之一,而变异链球菌是人类的主要致龋菌,它通过在牙齿表面的黏附和聚集形成生物膜,进而产酸耐酸致龋。因此研究其生长特性及生物膜结构能更好的研究龋病发病机理。近年来,随着科学技术的不断进步,对生物膜的研究也越来越深入。激光共聚焦显微镜与传统的电镜相比,前者不但可以保持生物膜的完整结构,而且可沿水平、垂直方向断层扫描,进行三维重建,使人们可以从不同的角度来分析生物膜的结构特性。但是由于其样本需经荧光标记或染色,使实验时间明显延长,于是报告基因技术用于细菌形态学研究的优势便得以体现。细菌通过转入的荧光蛋白自身发光,不需染色就可在镜下动态实时的观察。 本实验比较研究变异链球菌荧光报告株与野生菌株的生长能力和生物膜形成能力,结果显示二者无明显差异。随后研究在激光共聚焦显微镜下,报告株在树脂,玻璃离子表面形成生物膜的差异,为临床修复材料的选择提供了依据。本研究共包括三个实验。 实验一变异链球菌荧光报告株与野生菌株生长能力的比较 将复苏后的变异链球菌,变异链球菌荧光报告株调至相同的光密度值,37℃厌氧箱培养。于0,1.5,3,4,6,8,10,12,16,20,24h取培养液,酶标仪测OD600值。绘制生长曲线,结果两者生长趋势相近,提示两者具有相同的生长状态。 实验二变异链球菌荧光报告株与野生菌株生物膜形成能力的比较 在96孔细胞培养板中培养变异链球菌荧光报告株及野生株,分别于培养后4h, 8h, 12h, 24h取样,将生物膜用结晶紫染色,无水乙醇脱色,酶标仪测OD575。组间比较发现两菌株生物膜形成量无统计学意义(p0.05)。 在盖玻片上培养上述二菌,分别培养4h,8h, 12h,24h取样,通过洗涤、固定、脱水、干燥和喷金处理后,SEM下观察。结果发现不同时间段两者的生物膜形态没有明显差异,都是由早期散在分布,少量交联;到后期成团、成膜分布、各个菌落融合并重叠,形成网状结构。高倍镜下细菌形态也未见改变。提示两者生物膜形态相似。 实验三变异链球菌荧光报告株在不同材料表面形成生物膜能力的研究 在已制作好的树脂、玻璃离子片上培养变异链球菌荧光报告株,分别于培养后4h,8h, 12h,24h取样、洗涤、晾干、封片,激光共聚焦显微镜下观察,并在不同位置沿Z轴进行扫描,测量其厚度和组织结构。结果发现不同时间段玻璃离子表面形成的生物膜量及其厚度均少于同一时间段树脂表面,提示变异链球菌荧光报告株在玻璃离子表面形成生物膜的能力低于树脂表面。
[Abstract]:Dental caries is one of the common oral infectious diseases, and Streptococcus mutans is the main cariogenic bacteria. Therefore, the study of its growth characteristics and biofilm structure can better study the pathogenesis of caries. In recent years, with the development of science and technology, the study of biofilm is more and more in-depth. Compared with traditional electron microscope, laser confocal microscope can not only maintain the complete structure of biofilm, but also scan and reconstruct the biofilm in horizontal and vertical directions. It makes it possible to analyze the structure of biofilm from different angles. However, due to the fluorescence labeling or staining of the samples, the experimental time is obviously prolonged, so the advantage of the reporter gene technique in the study of bacterial morphology can be demonstrated. Bacteria can be observed dynamically and in real-time without staining through the fluorescent protein itself. The growth ability and biofilm forming ability of Streptococcus mutans fluorescent report strain and wild strain were studied in this experiment. The results showed that there was no significant difference between them. Then the difference of biofilm formation on the surface of resin and glass ion was studied under laser confocal microscope, which provided the basis for the selection of clinical repair materials. This study consists of three experiments. Experiment 1 comparison of growth ability between Streptococcus mutans fluorescent report strain and wild strain, the resuscitated Streptococcus mutans and Streptococcus mutans fluorescent report strain were adjusted to the same optical density value of 37 鈩，

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