【作者】 刘娅玲；

【导师】 周学东；

【作者基本信息】 四川大学 ， 口腔临床医学， 2006， 博士

【摘要】 从唾液和龈沟液中持续分泌的尿素被口腔细菌尿素酶快速分解产生氨和二氧化碳。尿素水解的碱性产物通过中和细菌产酸以维持牙菌斑的中性pH值环境，有助于稳定牙菌斑菌群平衡，防止牙釉质脱矿和促进牙釉质再矿化。因此，尿素水解被认为对防止龋病发生起到了重要作用。迄今为止，关于牙菌斑中哪些细菌具有尿素酶活性以及哪些细菌决定牙菌斑尿素酶活性的问题尚不清楚，对口腔细菌尿素酶基因表达的调控机制缺乏认识。本研究首先对牙菌斑中常驻细菌进行尿素酶活性检测，比较细菌间尿素酶的酶促动力学特征，寻找决定牙菌斑尿素酶活性的关键细菌。在此基础上，以内氏放线菌作为研究对象，了解细菌尿素代谢的生物学特征，模拟内氏放线菌在牙菌斑中的生物膜状态，探索环境因素和生存状态对细菌尿素酶活性和尿素酶基因表达的影响。 方法 采用连续培养的恒化器模型培养内氏放线菌单菌种生物膜，使用酶底物反应法和Real-time PCR技术分别在生化水平和基因水平检测氮源物质、糖源物质、pH值和清除率对尿素酶活性和尿素酶UreC基因表达的影响，同时比较细菌在膜状态下和浮游状态下尿素酶活性和尿素酶UreC基因表达的差异。 结果 研究发现，牙菌斑生物膜中细菌有活跃的尿素代谢活动，唾液链球菌，内氏放线菌能稳定表达尿素酶活性，血液链球菌和粘性放线菌尿素更多还原

【Abstract】 Urea are secreted from saliva and gingival crevicular fluid continuously and rapidly broken down by urease enzymes of oral microflora to ammonia and carbonhydrate. Alkali production from ureolysis could neutralize acid end products to stabilize pH homotasis in dental biofilms, so be helpful to stabilize microflora balance, inhibit enamel demineralization and promote mineral deposition. Thus, ureolysis is considered to be an important factor to prevent dental caries. High level of specific urease activity was detected in dental plaque, but understanding of the molecular characteristics of the urease enzymes of oral bacteria and the critical role that ureolysis play in ecological balance of dental biofilms are very limited. In this study, firsty, we concentrated on the examination of ureolytic bacteria in dental biofilms and compare the biochemical characteristics of the enzyme from different strains. Based on the result of the first part, we took Actinomyces neaslundii as research objective to study the physiological characteristics of ureolysis and effects of envirometal factors and growth state on the urease expression. Methods Continuous culture chemostat system was used to establish a nono-specie biofilms of A. neaslundii to study the effect of enviromental factors on the urease gene expression. In this part, biochemical methods and Real-time PCR techniques were used to detect the urease specific activity and urease mRNA levels as a function of pH, dilution rate, carbohydrate and nitrogen availability in fluid phase of culture medium. At the same time, the更多还原