【作者】 李果；

【导师】 吴红崑；

【作者基本信息】 四川大学 ， 口腔医学（专业学位）， 2022， 硕士

【摘要】 背景:根面龋是老年人常见口腔慢性病,发病率高、治疗率低、危害性大,严重影响老年人生活质量。口腔微生态失衡是根面龋发生发展的重要因素。课题组前期研究发现根面龋患者唾液中韦荣球菌属丰度显著增高。韦荣球菌属是口腔生物膜的早期定植菌属之一,其中小韦荣菌可促进链球菌生物膜形成,与龋病、牙周病、牙髓根尖周病等口腔疾病具有密切相关性。小韦荣菌在根面龋发生发展中的作用尚不清楚,其对根面龋相关微生物变异链球菌及白色念珠菌的影响及作用有待深入研究。目的:本研究通过分析老年根面龋患者的临床唾液样本,探究小韦荣菌与根面龋及变异链球菌、白色念珠菌的相关性,结合体外生物膜模型和大鼠根面龋模型,解析小韦荣菌对根面龋相关微生物变异链球菌和白色念珠菌致病毒力的影响与机制,为基于生物膜微生态平衡调节机制的根面龋病因学研究奠定理论基础,为根面龋的生态防治提供新的思路。方法:1、收集30名60岁及以上老年人中健康无龋者与患根面龋者的唾液,采用PCR以及q PCR检测小韦荣菌检出率和丰度值,探究小韦荣菌与根面龋发生的相关性;通过PCR以及q PCR检测唾液中根面龋致病相关微生物变异链球菌及白色念珠菌的检出率、丰度值,探究小韦荣菌与变异链球菌、白色念珠菌的相关关系。2、体外构建变异链球菌和白色念珠菌的单菌种生物膜、变异链球菌+白色念珠菌、小韦荣菌+变异链球菌、小韦荣菌+白色念珠菌的双菌种生物膜和变异链球菌+白色念珠菌+小韦荣菌的三菌种生物膜体外模型,利用结晶紫染色检测生物膜生物量,PCR检测生物膜中微生物组成,通过蒽酮-硫酸法、扫描电镜及激光共聚焦等技术观察生物膜结构及胞外多糖的合成,同时采用q RT-PCR检测变异链球菌及小韦荣菌相关毒力基因的表达,深入研究小韦荣菌对根面龋菌斑微生态的影响及对生物膜致龋毒力的调控机制。3、建立大鼠根面龋动物模型,将35只大鼠均分为7组:(1)空白对照组;(2)变异链球菌感染组;(3)白色念珠菌感染组;(4)变异链球菌+白色念珠菌感染组;(5)变异链球菌+小韦荣菌感染组;(6)白色念珠菌+小韦荣菌感染组;(7)变异链球菌+白色念珠菌+小韦荣菌感染组。给予致龋性饮食,采取电刀切龈暴露牙根后口腔涂抹微生物的方式构建不同微生物感染根面龋的疾病模型。七周后,收集大鼠唾液及上下颌颌骨样本,通过Doff’s根面龋评分法统计根面龋发病率、q PCR技术分析大鼠唾液样本中变异链球菌及白色念珠菌的丰度值,体内探究小韦荣菌介导根面龋相关微生物毒力在根面龋发病中的作用。结果:1、与健康无龋组相比,小韦荣菌在患根面龋组的唾液样本中检出率较高,具有统计学差异。小韦荣菌、变异链球菌及白色念珠菌在根面龋组唾液中富集程度显著高于根面无龋组,并且小韦荣菌丰度值与变异链球菌及白色念珠菌丰度值呈正相关性。2、分析体外生物膜模型,发现小韦荣菌定植可显著增加生物膜中变异链球菌生物量,促进生物膜胞外多糖的合成与分泌、形成更致密的生物膜,增强生物膜抗酸、耐氧等应激能力和对氯己定的抵抗能力,上调变异链球菌合成胞外多糖的gtf B、gtf C基因及产酸相关基因ldh的表达。小韦荣菌定植对生物膜中白色念珠菌生物量及毒力无显著影响。双菌种生物膜中,小韦荣菌过氧化氢酶合成的相关基因cat A显著上调。3、在大鼠根面龋模型中,小韦荣菌感染可影响大鼠唾液中的微生物组成,显著增加唾液中变异链球菌丰度值,提高大鼠的根面患龋指数结论:小韦荣菌与根面龋患病率具有重要相关性,可上调根面龋微生态中变异链球菌的丰度,促进胞外多糖合成,介导形成更致密的生物膜,增强生物膜抗胁迫能力,增加毒力基因表达,诱导或加速根面龋发生发展。本研究从牙菌斑微生态平衡的角度为根面龋病因学研究提供了理论依据,提示小韦荣菌是根面龋的潜在标识微生物之一,小韦荣菌与变异链球菌、白色念珠菌的临床检测有望协助根面龋的患病风险评估与疾病防治。更多还原

【Abstract】 Background: Root caries is a common oral chronic disease of the elderly,with high incidence,low treatment rate and great harm,which seriously affects the quality of life of the elderly.Oral microecological imbalance is an important factor in the development of root caries.The previous research of the research group found that the abundance of Veillonella in the saliva of patients with root face caries was significantly increased.Veillonella is one of the early colonizing bacteria of oral biofilm,of which Veillonella parvula can promote the formation of streptococcus biofilm,which is closely related to oral diseases such as caries,periodontal disease,and pulp apical periapical disease.The role of Veillonella parvula in the development of root caries is unclear,and its effect and mechanism on Streptococcus mutans and Candida albicans,which are related to the microorganisms associated with root caries,need to be studied in depth.Objective: In this study,through clinical sample analysis,combined with in vitro biofilm model and rat root caries model,this study aims to explore the correlation between Veillonella parvula and root caries,analyze the effect and mechanism of Veillonella parvula variant Streptococcus mutans and Candida albicans virulence,lay a theoretical foundation for the study of root caries based on the microecological balance regulation mechanism of biofilm,and provide new ideas for the ecological prevention and control of root cariesMethod:1.Collect the saliva of caries-free people and patients with root caries,detect the detection rate and abundance value of Veillonella parvula by PCR and q PCR,and explore the correlation between Veillonella parvula and the occurrence of root caries;exploring the detection rate and abundance values of Streptococcus mutans and Candida albicans in saliva by PCR and q PCR,to explore the correlation between Veillonella parvula variants and Streptococcus mutans and Candida albicans in saliva samples,and provide clinical basis for the correlation between Veillonella parvula and root caries and its related microorganisms.2.Construct the biofilm of Streptococcus mutans and Candida albicans monobacterium,Streptococcus mutans+ Candida albicans,Veillonella parvula +Streptococcus mutans,Veillonella parvula + Candida albicans and Streptococcus mutans+ Candida albicans + Veillonella parvula microbial biofilm in vitro,use crystal violet staining to detect biofilm biomass,observe the structure of biofilm and the synthesis of extracellular polysaccharides by anthracite-sulfuric acid method,scanning electron microscopy and laser confocal,and detect the microbial composition in the biofilm by PCR.At the same time,q RT-PCR was used to detect the expression of microbial-related virulence genes about Streptococcus mutans and Veillonella parvula,and the influence of Veillonella parvula on the microecology of root caries and the regulation mechanism of biofilm caries.3.Constructed a model of rat root caries,and divided 35 rats into 7 groups:(1)blank control group;(2)Streptococcus mutans infection group;(3)Candida albicans infection group;(4)Streptococcus mutans+ Candida albicans infection group;(5)Streptococcus mutans+ Veillonella parvula infection group;(6)Candida albicans +Veillonella parvula infection group:(7)Streptococcus mutans+ Veillonella parvula+Candida albicans infection group.A carious diet is given to construct a disease model of different microorganisms infecting the root caries by electro-knife cutting the gums and then applying microorganisms to the mouth after exposing the root of the tooth.After seven weeks,samples of rat saliva and upper and lower jaws were collected.The incidence of root caries was calculated by Doff’s root caries index method,and the abundance values of Streptococcus mutans and Candida albicans in rat saliva samples were analyzed by q PCR technology,and the role of Veillonella parvula in mediating the virulence of root caries-related microorganisms in the pathogenesis of roo caries was explored in vivo.Results:1.Compared with the caries-free group,The detection rate of Veillonella parvula in the saliva samples of the root caries group was higher,and there was a statistical difference.The concentration of Veillonella parvula,Streptococcus mutans and Candida albicans in saliva was significantly higher than those of the caries-free group,and the abundance values of Veillonella parvula were positively correlated with the abundance values of Streptococcus mutans and Candida albicans.2.Through the construction and analysis of in vitro biofilm model,it was found that the colonization of Veillonella parvula could significantly increase the biomass of Streptococcus mutans in the biofilm,promote the synthesis and secretion of extracellular polysaccharides in biofilm,form a denser biofilm,enhance the resistance of biofilm to acid,oxygen stress and chlorhexidine sterilization,and upregulate the expression of extracellular polysaccharide gtf B/gtf C and acidproducing related genes ldh of Streptococcus mutans.There was no significant effect on the biomass and virulence of Candida albicans in biofilms.In the bibacterial biofilm,the cat A peroxidase synthesis gene of Veillonella parvula was significantly upregulated.3.In the rat root surface carious model,the infection of Veillonella parvula can affect the microbial composition in the saliva of rats,significantly increase the abundance value of Streptococcus mutans in saliva,and make the root caries index of rats significantly higher than that of the control group.Conclusion:There is an important correlation between Veillonella parvula and root caries disease,which can promote extracellular polysaccharide synthesis by upregulating the abundance of Streptococcus mutans in the root caries microecology,mediate the formation of a denser biofilm,enhance the anti-stress ability of biofilms,upregulate virulence gene expression,and induce or accelerate the development of root caries.From the perspective of microecological balance of plaque,it provides a theoretical basis for the study of the etiology of root caries,and the research results suggest that Veillonella parvula is one of the potential identifying microorganisms of Veillonella and the clinical detection of Veillonella and Streptococcus mutans and Candida albicans is expected to assist in the risk assessment and disease prevention of rootfaced caries更多还原