【作者】 陈慧；

【导师】 赵立平；

【作者基本信息】 上海交通大学 ， 微生物学， 2016， 博士

【摘要】 口腔是消化道的起始部位,为微生物的黏附定植提供了许多不同的结合位点,是微生物在人体中重要的栖息环境。研究表明,微生物可以通过共聚集在口腔表面形成生物膜。在一个成熟的口腔生物膜系统中,微生物之间通过相互作用维持群落结构的稳定,并与宿主之间形成动态的生态平衡,为维持人体健康发挥重要的作用。当口腔环境由于不良的生活习惯等发生重大改变时,生物膜中的微生物群落结构发生失衡,一些条件致病菌大量增殖,反过来影响宿主的口腔健康甚至是全身健康。大量的临床研究表明,口腔微生物与口臭、龋齿和牙周病等口腔问题或疾病有着密切的关系,在糖尿病、心血管疾病等全身性系统疾病的发生发展中也可能起到十分重要的作用,与肺炎、慢性胃炎、结直肠癌等的发生也有一定的关系。研究不同疾病状态下口腔各部位菌群结构的变化,探究在疾病状态下微生物之间的相互关系,有助于更好地理解疾病的发生机制,为疾病的预防、诊断和治疗提供理论依据。本研究共分析了3种不同疾病状态下、不同口腔生境的微生物组组成特征。一、本研究采用16S rRNA基因焦磷酸测序技术分析了牙周炎患者和健康对照中唾液、龈上菌斑和龈下菌斑样本的微生物组成,发现不同口腔部位的菌群组成具有显著差异,而牙周炎患者和健康对照的口腔微生物组成也具有明显的区别。两步冗余分析(RDA)在唾液和龈上菌斑菌群中共挑选到21个共有的关键OTUs(Operational Taxonomic Units),与牙周炎以及牙周炎的两个重要的临床指标—牙周袋深度(pocket depth,PD)和附着丧失(attachment loss,AL)密切相关,包括Porphyromonas gingivalis,Tannerella forsythia和Filifactor alocis等。其中,Filifactor alocis与其它7个潜在致病菌之间存在较强的正相关(R>0.6,P<0.05),形成了一个以Filifactor alocis为中心、在口腔中共变化的微生物群,其丰度在牙周炎患者的三个口腔部位中显著升高,且对牙周炎具有较高的诊断价值。因此,本研究中找到的由8个共变化的牙周炎潜在致病菌组成的微生物群,各成员之间可能存在着一定的协同作用,共同促进牙周炎的发生发展。二、我们比较了33例根尖周炎患者(27例原发性根尖周炎、6例继发性根尖周炎)感染根管内的微生物组成,发现其主要由Bacteroidetes(47.2%)、Firmicutes(25.6%)、Synergistetes(6.3%)、Actinobacteria(5.7%)和Fusobacteria(5.3%)门的细菌组成,而Pseudomonas、Prevotella、Porphyromonas、Dialister和Fusobacterium这几个属的细菌则在根管内具有高检出率,是根尖周炎患者感染根管内的核心微生物类群。通过单变量统计分析,我们发现Tannerella和Streptococcus在继发性根尖周炎患者中的丰度显著高于原发性根尖周炎。此外,我们还发现Treponema maltophilum、Porphyromonas endodontalis和Solobacterium moorei的丰度分别在窦道、脓肿和疼痛组中显著升高,可能与这些临床症状相关,可以成为根尖周炎治疗的潜在目标,为个性化医疗提供理论基础。三、我们通过454焦磷酸测序和DGGE技术分析并比较了70名慢性胃炎患者和48名健康对照的舌苔微生物组成。研究发现,慢性胃炎患者的舌苔微生物组成与健康对照具有显著差异。通过冗余分析找到了52个在慢性胃炎患者和健康对照中存在显著差异的微生物类群。焦磷酸测序技术和DGGE分析都显示,Prevotella pallens、P.veroralis和Veillonella parvula/dispar在慢性胃炎患者的舌苔菌群中显著高于健康对照,可能与慢性胃炎的发生发展密切相关。相关分析的结果表明在疾病中显著升高的细菌更容易表现出较强的共变化趋势。进一步的研究表明,代表不同中医证型的白苔与黄苔、薄苔与厚苔的微生物组成未发现显著差异,而腻苔与非腻苔的微生物组成存在显著差异。多个临床指标表明,腻苔组病人的胃炎病情有较非腻苔组严重的趋势,我们发现有2个关键OTUs的丰度从健康对照到非腻苔组病人再到腻苔组病人逐渐显著增加,它们的最近邻居都为Megasphaera micronuciformis。这个细菌可能与慢性胃炎的病程有着密切的关系,值得进一步研究。综上,本研究通过高通量测序技术对牙周炎、根尖周炎以及慢性胃炎患者口腔特定部位的菌群结构进行了全面分析,涉及到唾液、龈上菌斑、龈下菌斑、根管以及舌苔这几个不同的口腔生境。综合三部分的研究发现,不同口腔生境的微生物组结构显著不同;但对于同一生境来说,比较疾病人群和健康对照在该生境的口腔微生物组结构,可以找到与疾病相关的特征性的菌群组成。本研究的结果有助于加深对疾病发病机制的理解,为疾病的干预提供了理论依据。更多还原

【Abstract】 Oral cavity is the initial part of the digestive tract,which offers many different binding sites for microbial adhesion and colonization,served as an important habitat for the microorganisms.It was reported that bacteria could co-aggregate on the surfaces of oral cavity and then form biofilms.In a mature oral biofilm,through the bacteria-bacteria interaction,the community structure maintains stable,and the inhabitants also show a dynamic ecological balance with the host,thus playing an import role to the body health.When the environment of the oral cavity changes,the balance of the microbial community structure will be destroyed and some opportunistic pathogens will be enriched,which may in turn influence the oral and even the whole body health of the host.Accumulated clinical studies have shown that the oral microbiota closely linked to several oral health problems or diseases,such as bad breath,caries and periodontal diseases,and may have relationship to the occurrence of several systemic system diseases like diabetes,cardiovascular disease.The oral microbiota may also play an important role in the development of pneumonia,chronic gastritis,colorectal cancer,etc.Exploring the structural changes of the oral microbiota in different oral habitats from different individuals under different disease states and digging the relationship between bacteria and diseased condition,will be helpful to better understanding the etiology of a disease and provide theoretical basis for the prevention,diagnosis and treatment of it.In the current research,three different works targeting three different human diseases using different oral samples were involved.First,we used 16 S rRNA gene pyrosequencing to profile the microbiota of saliva,supragingival and subgingival plaque from periodontitis patients and periodontally healthy volunteers.Different oral habitats harbored significantly different microbiota,and segregation of microbiota composition between periodontitis and health was observed as well.Two-step redundancy analysis identified twenty-one OTUs,including Porphyromonas gingivalis,Tannerella forsythia and Filifactor alocis,as potential pathogens that were significantly associated with periodontitis and two periodontitis diagnostic parameters(pocket depth and attachment loss)in both saliva and supragingival plaque habitats.Interestingly,pairwise correlation analysis among the 21 OTUs revealed that Filifactor alocis was positively correlated with seven other putative pathogens(R>0.6,P<0.05),forming a co-occurrence group that was remarkably enriched in all three habitats of periodontitis patients.This bacterial cluster showed a higher diagnostic value for periodontitis.Thus,our study identified a potential synergistic ecological pattern involving eight co-infecting pathogens across various oral habitats.Next,we compared the structure of microbiota in infected root canals obtained from thirty-three apical periodontitis patients,including 27 patients of primary infection and 6 patients of secondary infection.The results showed that Bacteroidetes(47.2%),Firmicutes(25.6%),Synergistetes(6.3%),Actinobacteria(5.7%)and Fusobacteria(5.3%)were the most predominant phyla in the microbiota of root canal.And several genera,including Pseudomonas,Prevotella,Porphyromonas,Dialister and Fusobacteria,showed high prevalence in infected root canals,thus could be regarded as the core taxa of the microbiota.As revealed by univariate statistical analysis,we found Tannerella and Streptococcus displayed a higher abundance in the root canal of the secondary infection than that of the primary infection.In addition,our work also revealed Treponema maltophilum,Porphyromonas endodontalis and Solobacterium moorei were significantly enriched in individuals with sinus tract,swelling or pain,respectively,indicating that these bacteria may be associated with the different clinical symptoms of apical periodontitis,thus might be potential targets for the disease treatment.We also investigated the tongue coating microbiota of 70 gastritis patients and 48 healthy controls through pyrosequencing and DGGE.Distinct segregation of the microbiota between the gastritis and the healthy was observed by both methods.Redundancy analysis identified 52 OTUs which were significantly different between those two groups.Particularly,Prevotella pallens,P.veroralis and Veillonella parvula/disparas were significantly enriched in the tongue coating microbiota of patients compared with those of healthy ones,thus were regarded as the key phylotypes associated with gastritis.Correlation analysis revealed the gastritis-enriched phylotypes showed more remarkable co-abundant behavior.Further studies revealed neither the tongue coating microbiota between white and yellow fur nor those between thin and thick fur,the different tongue appearance representing the different types of TCM syndromes,were significantly different.This implied the relationship between tongue coating microbiota and TCM syndromes is still unclear.However,the microbiota between greasy fur and non-greasy fur,two types of gastritis patients seeming to have different degree of the disease,showed significant difference with each other.Two OTUs,which were both closely to Megasphaera micronuciformis,increased their relative abundance in patients compared with healthy controls,and showed the highest abundance in patients with greasy fur.This bacterium might be the critical organism associated with the development of gastritis and worth to be further studied.In conclusion,the oral microbiome structure in diverse oral habitats(including saliva,supragingival plaque,subgingival plaque,root canal and tongue coating)form periodontitis,apical periodontitis and chronic gastritis patients were investigated comprehensively through high-throughput sequencing technologies.Integrating the data from the three studies,we found the oral microbiome in different oral habitats were significantly different.For a certain oral habitat,the characteristic disease-associated communities can be found by comparing the microbiome in this oral habitat between diseased and healthy cohorts.The results of this study will help to deepen the understanding of disease pathogenesis and provide theory basis for the intervention of the disease.更多还原