唾液乳杆菌对变异链球菌生物膜形成的抑制机制

Dental caries is common oral bacterial infectious disease. Its prevention and treatment requires control the biofilm formation of causative pathogens, such as Streptococcus mutans. Some probiotic bacterias, such as Lactobacillus, are also known to possess positive effects on biofilm formation. Lactobacillus salivarius REN (L. salivarius REN) was shown to have several functions, such as inhibition of Streptococcus mutans, oral cancer prevention, the mechanism remains unknown. In this study, the model of Saliva-coated Hydroxyapatite (S-HA) will be used to demonstrate the adhesion mechanism of L. salivarius REN and salivary pellicles. The adhesion and receptors will be identified by SLP-null mutants, Pull-down and co-immunoprecipitation assay firstly. Then, a two-species biofilm formed on S-HA discs will be used to demonstrate the adhesion capability of S. mutans to S-HA before or after presence of L. salivarius REN. Biofilm mass, levels of calcium depletion, acidity, structure of biofilm will be determined. In order to clarify the inhibitory mechanism, quantitative real-time polymerase chain reaction and Western Blot will be used to detect the expression of genes encoding quorum-sensing signals and glucosyltransferases. This project will provide direct scientific evidence for inhibition mechanism of L. salivarius on adhesion and biofilm formation of S. mutans, elucidate the relationship between the lactobacillus and dental caries.

变异链球菌生物膜是龋病发生的主要因素，如何抑制其形成是龋病预防的核心。乳酸菌能抑制变异链球菌的牙面黏附和生物膜形成，作用机制尚不明确。项目以唾液乳杆菌REN（L. salivarius REN）为对象，以唾液包被羟磷灰石模拟牙环境，首先比较表面蛋白缺失突变株和野生株的黏附能力，筛选表面黏附素；利用Pull-Down、免疫共沉淀等技术筛选L. salivarius REN与唾液获得性膜的黏附受体，阐明黏附机制；以L. salivarius REN与变异链球菌共培养的物膜为对象，利用Western Blot、RT-PCR及扫描电镜等技术，验证L. salivarius REN对生物膜的抑制作用，寻找L. salivarius REN对变异链球菌LuxS/AI-2系统和糖基转移酶的调控规律，阐明抑制机理。研究将揭示唾液乳杆菌竞争性黏附和抑制生物膜形成的作用机制，也为其在龋病预防领域应用提供支持。

对口腔中致病菌的控制和预防黏附是对预防口腔及其他系统疾病具有至关重要的意义。本课题基于实时荧光定量PCR技术和高通量测序技术，从体外羟磷灰石模型模拟牙齿表面、小鼠龋齿模型和人群口腔试验三个层面探讨副干酪乳杆菌L9对口腔致病菌的抑制黏附作用，并监控副干酪乳杆菌对口腔菌群的调节作用，从而为预防和控制口腔疾病提供有效措施和理论依据。.本文通过在体外构建口腔模型，建立了基于RT-PCR技术的致病菌的定量检测方法，并且证明副干酪乳杆菌L9显著抑制了戈氏链球菌和变异链球菌在羟磷灰石上的黏附，在培养2、6、18h后，戈氏链球菌黏附量分别降低了51.19％、56.09％和58.40％、变异链球菌降低了26.88％、33.26％和42.25％，同时副干酪乳杆菌L9显著减少了变异链球菌在羟磷灰石上形成的生物膜的厚度和密度。.利用变异链球菌诱导小鼠龋齿模型，探究副干酪乳杆菌L9对小鼠口腔龋齿的预防和对口腔菌群的调节作用，结果显示：与模型组相比，L9组显著缓解咬合面E级龋齿的发生程度；通过多种致龋因子的RT-PCR的分析，表明L9的预防龋齿功能与其抑制变异链球菌的初始黏附和牙菌斑生物膜的发展过程中的蔗糖依赖性黏附有关；口腔菌群的16srDNA高通量测序结果表明，模型组口腔菌群多样性指数无显著差异，但菌群结构变化显著，尤其是放线菌门显著上调，而L9组的放线菌门明显下调，且在属分类水平上的克雷伯菌属、气球菌属、Gemella、棒状杆菌和铜绿假单胞菌显著下调，降低了口腔疾病的风险。.通过高通量测序的方法检测L9对正常人群唾液和牙菌斑菌群的影响，并同时检测唾液pH和口腔中VSC含量的变化情况。结果显示，两周食用L9益生菌含片后，唾液物种丰富度显著下调，链球菌科、放线菌科、消化链球菌科等口腔疾病相关菌的相对丰度显著下调；牙菌斑菌群物种多样性显著下调，纤毛菌科、梭杆菌科及黄杆菌科的相对丰度均显著降低。.综上，副干酪乳杆菌L9能够通过减少口腔致病菌的初始黏附和牙菌斑形成，使口腔菌群得到调节作用，减少口腔疾病的发生。