重庆市6-8岁儿童龋病相关口腔微生物组及唾液蛋白组分析

发布时间：2018-07-13 17:06

【摘要】：研究背景：龋病是一种发病率高、缺乏自限性的感染性疾病，可影响颌面部美观、功能及全身影响水平。就龋病的控制来说，我们应当坚持预防为根本，治疗是手段的策略。而龋病的预防，关键在于对各种龋病危险因素的控制。细菌因素（牙菌斑）是龋病发生的始动因素，而致龋相关的口腔微生物组结构十分复杂。本课题组前期采用基因芯片及454焦磷酸测序对不同人群龋易感儿童的牙菌斑微生物进行了系列研究，其结果进一步凸显了微生物致龋的复杂性，微生物与龋病的关系尚需进一步深化认识。当前，“核心微生物组”是一个热门的研究课题。多数研究表明口腔中可能存在一个“核心微生物组”,但尚有学者持不同观点。本课题组前期研究支持口腔“核心微生物组”存在的可能性，本实验拟在前期基础上，进一步为口腔“核心微生物组”是否存在提供一定实验依据并探索其可能的微生物组结构。除了口腔细菌与龋病息息相关，唾液蛋白组也与龋病关系密切。同时，唾液蛋白组是潜在的诊断和预防局部和系统性疾病的生物标记物来源之一。系统探索唾液蛋白组和口腔微生物组多样性将有助于对龋病等口腔局部疾患和系统疾病的认识、预防和诊疗。目的：分析比较高龋组和无龋组儿童牙菌斑（唾液）微生物组结构多样性以及高龋组和无龋组儿童的唾液蛋白组。第一部分从不同分类学水平（如门、属等）比较高龋组和无龋组的微生物组结构组成和差异，并探索“口腔核心微生物组”是否存在。第二部分探索唾液蛋白组与龋病的关系，并推动龋易感性相关生物标记物的研究。方法：以6-8岁学龄儿童为研究对象，使用454焦磷酸测序分析比较高龋组和无龋组牙菌斑（唾液）微生物组结构和种类（实验一），采用高压液相柱色谱—串联质谱分析及对比高龋组与无龋组唾液蛋白组（实验二）。结果：实验一：（1）本实验平均每个样本检出超过6000个OTU，总共得到14076个OTU，归于18个门，，28纲,48目,78科,133个属。其中高龋组含9265个OTU（高龋菌斑6042个OTU，高龋唾液5406个OTU），归属于13个门，111种属；无龋组含10080个OTU（无龋菌斑6757个OTU，无龋唾液5661个OTU），归属于17个门,111种属。（2）总共有15个菌属在高龋菌斑组与无龋菌斑组之间存在有统计学意义的差异（p0.05），其中5个菌属在高龋菌斑组的检出高于无龋菌斑组，包括放线菌属、莫拉克斯氏菌属、史雷克氏菌属等；10个菌属在无龋菌斑组检出率高于高龋菌斑组，包括伯杰氏菌属、二氧化碳嗜纤维菌属、心杆菌属、德克斯氏菌属、颗粒链菌属、嗜血菌属、金氏菌属、巨型球菌属、奈瑟氏菌属等；（3）总共17个菌属在高龋唾液组与无龋唾液组之间存在有统计学意义的差异（p0.05），其中11个菌属在高龋唾液组的检出高于无龋唾液组，包括放线杆状菌属、丁酸弧菌属、德克斯氏菌属、戴阿利斯特杆菌属、乳杆菌属、丙酸弧菌属、假支杆菌属、链杆菌属等；6个菌属在无龋唾液组检出率高于高龋唾液组，包括卟啉单胞菌属、嗜血菌属、不动杆菌属、小链菌属、戴尔福特菌属等；（4）公认致龋菌如变形链球菌、内氏放线菌、乳杆菌在各样本中检出率较低，在高龋组、无龋组之无明显差异；（5）10个高龋菌斑样本与10个无龋菌斑样本共有27个菌属，包括：放线菌属、伯杰氏菌属、二氧化碳嗜纤维菌属、毛单胞菌属、棒状杆菌属、德克斯氏菌属、梭杆菌属、金氏菌属、孪生菌属、颗粒链菌属、约翰森氏菌属、纤毛菌属、奈瑟氏菌属、卟啉单胞菌属、普氏菌属、链球菌属、密螺旋体属、韦荣菌属等；10个高龋唾液样本与10个无龋唾液样本共有24个菌属，包括：放线菌属、伯杰氏菌属、弯曲杆菌属、二氧化碳嗜纤维菌属、棒状杆菌属、德克斯氏菌属、梭杆菌属、孪生菌属、颗粒链菌属、约翰森氏菌属、纤毛菌属、奈瑟氏菌属、消化链球菌属、卟啉单胞菌属、普氏菌属、丙酸菌属、罗氏菌属、月形单胞菌属、链球菌属、韦荣菌属等。实验二：（1）本实验有定量信息的人蛋白质1626个，从高龋组及无龋组唾液样本中共检出642个差异表达蛋白，包含基质金属蛋白酶9、粘蛋白MUC7、乳铁蛋白、碳酸酐酶6、天青杀素、冷凝集素等可能与龋病密切相关的蛋白；（2）本实验鉴定出的蛋白GO分析提示：蛋白质分子功能中，最多的是蛋白结合性（28.82%），其次是催化活性（22.58%），然后是金属结合性（8.74%）及核酸结合（8.58%）；就参与生物学过程来说，参与代谢过程的蛋白质较多（19.3%），其次为调节过程（12.39%）。结论：（1）本课题组大部分相关实验均发现韦荣菌属在高龋菌斑组检出高于无龋菌斑组，其存在致龋性可能性较高，具体作用有待进一步研究求证；（2）菌斑二氧化碳嗜纤维菌属等与龋病呈负相关关系，唾液二氧化碳嗜纤维菌属与龋病呈正相关关系，其构成比可作为潜在的监测龋易感性的指标；（3）牙菌斑中多种细菌参与了龋病过程（如韦荣菌属、放线菌属、莫拉克斯氏菌属、史雷克氏菌属等），可不需要大量变形链球菌、乳杆菌等公认致龋菌定植，龋病是多种菌斑微生物共同作用的结果，支持“菌斑微生态学说”；（4）放线菌属、伯杰氏菌属、二氧化碳嗜纤维菌属、梭杆菌属、孪生菌属、奈瑟氏菌属、卟啉单胞菌属、普氏菌属、链球菌属、韦荣菌属等可能是“核心微生物组”的一部分，支持口腔“核心微生物组”的存在；（5）本研究初步表明高龋组儿童唾液蛋白总量及种类均高于无龋组， MMP9、MUC7、LTF、CA6、AZU、冷凝集素等高龋组与无龋组之间的差异表达蛋白，可能为进一步探索龋易感性相关生物标记物提供了一定的实验依据。
[Abstract]:Background: caries are a high incidence and lack of self limiting infection, which can affect the aesthetic, functional and systemic effects of the maxillofacial region. As to the control of caries, we should adhere to the strategy of prevention as the fundamental and treatment means. The prevention of caries is the key to the control of various caries risk factors. Plaque) is the starting factor of caries occurrence, and the structure of dental caries related microorganism is very complex. In our group, a series of studies were carried out by gene chip and 454 pyrophosphate sequencing for dental plaque microbes of children with caries susceptibility in different populations. The results further highlighted the complexity of microbial caries, microorganism and caries. The relationship needs to be further deepened.
At present, the "core microorganism group" is a hot research topic. Most studies have shown that there may be a "core microorganism group" in the oral cavity, but some scholars hold different views. The previous study of this group supports the possibility of the existence of the oral core microorganism group. Whether the heart microorganism group exists can provide some experimental evidence and explore its possible microbial structure.
In addition to dental caries, the sialin group is also closely related to caries. At the same time, the sialin group is one of the potential sources of biomarkers for the diagnosis and prevention of local and systemic diseases. The systematic exploration of the diversity of the salivary protein group and the oral microorganism group will contribute to dental caries and systemic diseases, such as dental caries. Understanding, prevention and diagnosis and treatment.
Objective: To compare the structural diversity of dental plaque (saliva) of children in high caries group and non caries group, and the sialin group of children in high caries and non caries groups. The first part compared the structure and difference of microbiological groups in high caries and caries free groups from different taxonomic levels (such as doors, genera, etc.), and explored the "oral core microorganism group". The second part is to explore the relationship between salivary proteome and caries, and to promote the study of biomarkers of caries susceptibility.
Methods: 6-8 year old school-age children were studied by using 454 pyrosequencing analysis to compare the structure and type of dental plaque (saliva) group of high caries group and non caries group (experiment one). High pressure liquid chromatography tandem mass spectrometry was used to analyze and compare the sialin group of high caries group and no caries group (experiment two).
Result:
Experiment 1: (1) in this experiment, the average samples were detected more than 6000 OTU in each sample, with a total of 14076 OTU, belonging to 18 gates, 28 classes, 48 orders, 78 families, 133 genera. Among them, the high caries group had 9265 OTU (high caries plaque 6042 OTU, high caries saliva 5406 OTU), belonging to 13 gates, 111 species, and caries free group containing OTU, no caries saliva. 61 OTU), belonging to 17 doors and 111 genera. (2) there were a total of 15 bacteria in the high caries plaque group and the non caries plaque group (P0.05), of which 5 bacteria in the high caries plaque group were higher than the non caries plaque group, including the actinomycetes, Mora KSR, srek, and so on; 10 bacteria in the caries free bacteria. The detection rate of the plaque group was higher than that of the high caries plaque group, including Berger, carbon dioxide, cardiobacteria, Dekkers S, Haemophilus, Haemophilus, aureus, mega, and Neisseria. (3) a total of 17 bacteria were statistically different between the high caries saliva group and the caries free saliva group (P0.05 11 bacteria in the high caries saliva group were higher than those in the non caries saliva group, including the actinomycetes, Vibrio butyrate, Dekkers S, Lactobacillus, Lactobacillus, Vibrio propionate, pseudobranched and chain bacilli, and the 6 bacteria in the caries free saliva group were higher than the high caries saliva group, including Porphyromonas. Haemophilus, Acinetobacter, small Streptococcus, DELL, Ford, etc.; (4) it is recognized that cariogenic bacteria such as Streptococcus mutans, actinomycetes, Lactobacillus in each sample are low, in high caries group, no caries group has no obvious difference; (5) 10 high caries plaque samples and 10 non caries plaque samples altogether have 27 bacteria, including: actinomycetes, Bo Genera, carbon dioxide, Corynebacterium, Corynebacterium, Corynebacterium, Clostridium, Corynebacterium, twin, pellevibrium, Johannsen, cilium, Neisseria, porphyrinomila,, Streptococcus, Treponema, Wei Rong, and so on; 10 high caries saliva samples There are 24 genera and 10 caries free saliva samples, including actinomycetes, Berger, Campylobacter, Corynebacterium, Corynebacterium, Corynebacterium, Clostridium, twin, granular, Johannsen, cilium, Neisseria, Streptococcus, porphyrinomonas, and prissiella Genus Propionibacterium, rhotococcus, monomonas, Streptococcus, vyjuna, etc.
Experiment two: (1) 1626 human proteins with quantitative information were found in this experiment. 642 differentially expressed proteins were detected from the high caries group and the caries free group, including the matrix metalloproteinase 9, mucin MUC7, lactoferrin, carbonic anhydrase 6, azidotin, cold agglutinin and other proteins that could be closely related to caries; (2) the eggs identified by this experiment White GO analysis suggests that protein binding is the most important protein binding (28.82%), followed by catalytic activity (22.58%), then metal binding (8.74%) and nucleic acid binding (8.58%); in biological processes, more proteins (19.3%) are involved in the metabolic process (19.3%), followed by the regulation process (12.39%).
Conclusions: (1) most of the related experiments in our group found that the detection of the genus weihonia in the high caries plaque group was higher than that of the non caries plaque group, and the possibility of its cariogenic possibility was higher. (2) the plaque carbon dioxide eosinophilia was negatively related to caries, and the saliva carbon dioxide eosinophilia and caries were in the form of caries. Positive correlation, its composition can be used as a potential index for monitoring susceptibility to caries; (3) many bacteria in dental plaque are involved in caries process (such as Vibrio, actinomycetes, Morach J S, sphacella, etc.), which do not require a large number of Streptococcus mutans, Lactobacillus, and other recognized caries colonies, and the caries are a variety of microbiological plaque. The results of the common action support the "microecological theory of plaque"; (4) the genus actinomycetes, Berger, carbon dioxide, Clostridium, twinning, Neisseria, porphyrinomonas, prissiomycetes, Streptococcus, and Vibrio may be part of the "core microorganism", supporting the "core microbes" in oral cavity. 5. (5) this study showed that the total amount and type of saliva protein in the high caries group were higher than those in the non caries group. The differential expression proteins between the high caries group and the caries free group such as MMP9, MUC7, LTF, CA6, AZU and cold agglutinin may provide a certain experimental basis for further exploring the biomarkers of caries susceptibility related products.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R788.1

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