密度感应信号系统luxS/AI-2在持续性根尖周炎根尖生物膜形成中的作用研究

One of the mechanisms by which microorganisms interact with each other is quorum sensing (QS) which has been identified in both Gram-negative and Gram-positive species of bacteria and is used to regulate diverse functions, such as biofilm formation. Rencently, it is found that the luxS mediated quorum sensing (AI-2 signalling) plays an important role in interspecies communication and has been implicated in virulence and persistence infections,and biofilms formation.It is generally acknowledged that the main reason for the persistent apical periodontitis is persistent intraradicular and extraradicular infections. Previously, we evaluated the prevalence of bacteria in apical biofilm and periradicular lesion associated with persisitent apical periodontitis using 16SrDNA clone libraries and examined apical biofilm morphologic structure using the SEM and CLSM. The results suggested that apical biofilm is the main source of persistent apical infection and E. faecalis，S.intermedius，P.gingivalis and F. nucleate were predominant in apical biofilm. Bacteria organized in biofilms are protected from host defenses and antibiotics and can be the source of persistent infections that are very difficult to eradicate. On these grounds, hypothesis was proposed that the LuxS/AI-2 signaling system plays an important role in formation of apical biofilm assosiated with persistent periapical periodontitis. In order to prove the relationship between LuxS/AI-2 signaling system and formation of apical biofilm, through constructing dominant bacteria biofilm models to compare different dominant bacteria ability of biofilm formation in an effort to contribute to the ongoing characterization of correlation between dominant bacteria and apical biofilm. The luxS gene was detected using PCR..Then, the luxS deletion mutant strains were constructed using homologous recombination and their biofilm formation. Compared to the standard strain, morphous and struction of biofilm and expression of adherence factors in the luxS deletion mutant strains were evaluated. These results suggest that luxS may be involved in the interruption of bacterial communication and apical biofilm formation that contribute to the virulence of the bacterium. A thorough knowledge of LuxS/AI-2 signaling system and formation of apical biofilm could guide new strategies to combat infection, leading to a better prognosis for root-canal re-treatments.

密度感应信号系统luxS/AI-2是细菌之间交流的主要渠道，可以调节多种细菌生物膜的形成，与持续感染密切相关。本课题组前期研究证实根尖生物膜是持续性根尖周炎的主要感染源，是导致根尖病难以愈合的原因。在根尖生物膜中检出粪肠球菌、中间链球菌、牙龈卟啉单胞菌和具核梭杆菌等几种优势菌。为进一步探讨根尖生物膜形成的机制，本研究采用分子生物学方法检测持续性根尖周炎根尖生物膜中luxS基因表达，通过体外构建优势菌生物膜模型，检测luxS基因和AI-2分子活性及形态特点，根据该结果构建luxS基因敲除突变株生物膜模型，与标准株作对比，采用实时定量PCR检测粘附相关基因表达水平， CLSM观察生物膜形成的结构特点，显微共焦拉曼光谱技术分析细菌和胞外基质的空间分布及分子构向，进而阐明luxS基因对优势菌根尖生物膜形成的调节作用。本研究有利于认识持续性根尖周炎的感染机制并为其治疗提供新思路。

密度感应信号系统luxS/AI-2是细菌之间交流的主要渠道，可以调节多种细菌生物膜的形成，与持续感染密切相关。本课题组前期研究证实根尖生物膜是持续性根尖周炎的主要感染源，是导致根尖病难以愈合的原因。在根尖生物膜中检出粪肠球菌、中间链球菌、牙龈卟啉单胞菌和具核梭杆菌等几种优势菌。为进一步探讨根尖生物膜形成的机制，本研究采用分子生物学方法检测持续性根尖周炎根尖生物膜中luxS基因表达，通过体外构建优势菌生物膜模型，采用分子生物学方法检测luxS基因，根据该结果构建luxS基因敲除突变株生物膜模型，与标准株作对比，采用实时定量PCR检测粘附相关基因表达水平， CLSM观察生物膜形成的结构特点，进而阐明luxS基因对优势菌根尖生物膜形成的调节作用。本研究有利于认识持续性根尖周炎的感染机制并为其治疗提供新思路。. 本研究临床样本检测显示faecalis, P. gingivalis，及其LuxS 基因与PAP持续感染有密切关系。同时分离获得E.faecalis，P. gingivalis，F. nucleate菌株，为后续研究奠定坚实基础。通过形态学、分子生物学等方法显示E.faecalis具有较强的生物膜形成能力，密度感应系统LuxS基因表达水平与其生物膜形成能力有明显相关性。通过长臂同源多聚酶链反应技术构建E.faecalis 、P. gingivalis LuxS基因缺陷株成功。直接通过基因缺陷菌株证明LuxS基因是调控E.faecalis形成生物膜形成的关键基因，同时影响E.faecalis其他生物膜形成相关因子的表达。. 综上所述，密度感应信号系统与持续性根尖周炎的持续感染密切相关，未来该疾病的治疗应不仅限于手术清楚感染，在如何破坏密度感应系统从而控制感染角度应进行更深入研究。