不同患龋状态双胞胎儿童及其母亲龈上菌斑中微生物多样性分析

发布时间：2018-05-17 15:47

本文选题：双胞胎 + 龋病　；参考：《武汉大学》2017年硕士论文

【摘要】：龋病是学龄前儿童口腔内最常见的疾病之一。低龄儿童龋(EarlyChildhood Caries,ECC)是指71个月或更小的儿童口腔中任意一个乳牙存在一个或一个以上龋坏(包括已形成龋洞及白垩色龋损)、因龋充填,因龋缺失牙齿。全国第三次口腔流行病学调查结果表明,乳牙患龋率在5岁儿童高达66%,显著高于其他国家,表明在我国乳牙龋仍然是儿童口腔的一种常见病、多发病,现已成为一个重要的公众卫生问题,对儿童身体健康和生长发育都会产生较大的威胁,因而对龋病病因学进行深入细致地研究,最终找到合理的预防措施的任务仍相当艰巨。龋病病因的四联因素理论认为宿主、饮食、口腔中的微生物和时间共同参与了龋病的发生和发展,其中口腔微生物作为最重要的一个因素得到了口腔临床医生和科研工作者的广泛关注,虽然有很多研究关注宿主的全身健康、饮食习惯、环境因素等对龋病发生的作用,但更多的还是关注口腔中可培养的微生物诸如变形链球菌、乳杆菌、放线菌等在引起龋病发生过程中的作用,家庭环境和遗传因素对龋病发生的影响到目前为止还不是很明确。目的:本实验采用第二代人类口腔微生物芯片(Human Oral Microbe Identification using Next Generation Sequencing,HOMINGS)分析比较拥有不同患龋状态双胞胎儿童及其母亲口腔内龈上菌斑中菌群的结构及微生物多样性,从口腔细菌微生物层面和宿主遗传及环境因素方面初步探讨ECC的发病机制,为后期儿童龋病的预防、诊断与治疗提供一定的理论依据。方法:根据世界卫生组织(WHO)推荐的第4版龋病诊断标准,对符合本本实验条件的武汉市3-6岁双胞胎儿童及其母亲进行口腔健康检查,要求所有受试者无系统性全身性疾病,口腔健康检查前3个月内未服用过抗生素,无中度或重度牙周疾病及严重口腔黏膜疾病。选取符合条件14对双胞胎儿童(1人患龋1人无龋),1组三胞胎(2人患龋1人无龋)和15位母亲,要求有龋组(n=16)DMFIT≥2,无龋组(n=15)DMFT=0。牙菌斑样本取自14对双胞胎、1组三胞胎儿童(年龄3-6岁)和15位母亲,共46份样本,分别提取龈上菌斑细菌总DNA,送到福赛研究中心,采用HOMINGS分析龈上菌斑中菌群结构及微生物的多样性。结果:1.通过HOMINGS所使用的Miseq测序平台进行高通量测序共获得2,294,650条有效序列,使用QIIME软件在相似性大于97%的水平上聚类操作分类单元(Operational taxonomic unit,OTU),共得到 20,431 个 OTU,与 Greengene数据库中的参考序列进行比对和物种注释获得11个门,116个属和189个种水平的物种;2.在门水平上,母亲组在拟杆菌门和厚壁菌门的丰度显著高于有龋组和无龋组;3.在属水平上有龋组和无龋组比较,有龋组在链球菌属、乳杆菌属、放线菌属、嗜血杆菌的丰度要高于无龋组(P0.05);母亲组的微生物种类较儿童组多,分别在普氏菌属、坦纳氏菌属的相对丰度高于儿童组(P0.05);4.Alpha多样性分析结果表明母亲组龈上菌斑微生物组成的丰富程度和均匀程度显著高于有龋组和无龋组(P0.05);5.通过比较同卵和异卵双胞胎之间加权的进化距离(weighted unifrac distance),得到同卵双胞胎较异卵双胞胎龈上菌斑微生物组成更为相似。结论:1.链球菌属、乳杆菌属、放线菌属、嗜血杆菌属是与低龄儿童龋(ECC)显著相关的"核心微生物组";2.成年人口腔内龈上菌斑的微生物组成的丰富程度和均匀程度与儿童相比存在显著差异,说明在不同年龄阶段,口腔微生态环境是在发生变化的,而且成年人口腔内的微生物菌群结构使其更易患牙周疾病;3.同卵双胞胎较异卵双胞胎龈上菌斑的菌群组成更为相似说明遗传因素对口腔微生态的物种组成是有一定影响的。
[Abstract]:Dental caries are one of the most common oral diseases in preschool children. EarlyChildhood Caries (ECC) refers to the existence of one or more caries (including caries and Cretaceous caries) in children's oral cavity of 71 months or younger children. The third oral epidemiology of dental caries is the result of caries filling and dental caries. The results show that the dental caries rate of 5 year old children is up to 66%, which is significantly higher than that of other countries. It shows that dental caries in our country are still a common disease in children's mouth. It has become an important public health problem, and has a great threat to the health and growth of children, so the caries etiology is deep. In detail, the task of finding reasonable preventive measures is still quite arduous. The quadruple factor theory of the cause of caries considers that the host, diet, microorganism and time in the oral cavity participate in the occurrence and development of dental caries. Oral microorganism is one of the most important factors in oral clinicians and researchers. Widely concerned, although many studies have focused on the effects of host body health, dietary habits and environmental factors on dental caries, more attention is paid to the role of cultured microbes in oral cavity, such as Streptococcus mutans, Lactobacillus, actinomycetes, etc., in the process of dental caries, family environment and genetic factors. The effect is not clear so far. Objective: To compare the structure and microbial diversity of the bacteria in the oral gingival plaque of the twin children with different caries status and their mothers with second generations of Human Oral Microbe Identification using Next Generation Sequencing, HOMINGS. The pathogenesis of ECC is preliminarily discussed from the aspects of oral bacterial microorganism and host genetic and environmental factors, which provide a certain theoretical basis for the prevention, diagnosis and treatment of children's caries in the later period. Methods: according to the fourth version of the dental caries recommended by the WHO (WHO), the 3-6 year old double cell fetus in Wuhan City, which is in accordance with the original experimental conditions Children and their mothers were examined for oral health, requiring all subjects to have no systemic systemic disease, no antibiotics in the first 3 months of oral health examination, no moderate or severe periodontal disease and severe oral mucosal disease. 14 pairs of twins (1 with caries, 1 without caries), and 1 triplets (2 caries 1 without caries) were selected. 15 mothers were required to have a caries group (n=16) DMFIT > 2. The caries free group (n=15) DMFT=0. plaque samples were taken from 14 pairs of twins, 1 triplet children (age 3-6 years old) and 15 mothers, with 46 samples, respectively, to extract the total DNA of the bacteria on the gingival plaque, sent to the fusai Research Center, and used HOMINGS to analyze the flora structure and microbial diversity in the supra gingival plaque. Results: 1. a total of 2294650 effective sequences were obtained by high flux sequencing using the Miseq sequencing platform used by HOMINGS. Using QIIME software, a total of 20431 OTU, which were compared with the reference sequence in the Greengene database, was compared with the reference sequence in the Greengene database, and the Operational taxonomic unit, OTU was used in the level of similarity greater than 97%. The annotation obtained 11 gates, 116 genera and 189 species of species; 2. at the portal level, the abundance of the mother group in the bacteriobacterium and the thick wall bacteria was significantly higher than that in the caries and the caries free groups. 3. at the level of caries and non caries, the abundances of the caries, the lactobacillus, the actinomycetes, and the actinomycetes in the caries group were higher than those in the caries free group (P0.05 The microbial species in the mother group were more than those in the children's group, and the relative abundance of the genus tanabi was higher than that in the children's group (P0.05). The 4.Alpha diversity analysis showed that the abundance and uniformity of the microbial composition of the gingival plaque in the mother group was significantly higher than that in the caries and the caries free groups (P0.05); 5. by comparing the identical and heterogenous twins. The weighted evolutionary distance (weighted UniFrac distance) showed that identical twins were more similar to the microbiological composition of the supra gingival plaque in identical twins. Conclusion: 1. Streptococcus, Lactobacillus, actinomycetes, and Haemophilus is a "core microorganism group" that is significantly associated with low age children's caries (ECC); 2. adult oral gingival plaque The richness and uniformity of biological composition are significantly different from those of children, indicating that the microecological environment of the oral cavity is changing in different ages, and the microbial flora in the oral cavity of adults makes it more susceptible to periodontal disease, and 3. identical twins are more similar than those of the gingival plaque of the ISO twins. It indicates that genetic factors have certain effects on the species composition of oral microecology.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R788.1

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