【摘要】：研究背景：齲病是一種發(fā)病率高、缺乏自限性的感染性疾病，可影響頜面部美觀、功能及全身影響水平。就齲病的控制來說，我們應(yīng)當(dāng)堅(jiān)持預(yù)防為根本，治療是手段的策略。而齲病的預(yù)防，關(guān)鍵在于對(duì)各種齲病危險(xiǎn)因素的控制。細(xì)菌因素（牙菌斑）是齲病發(fā)生的始動(dòng)因素，而致齲相關(guān)的口腔微生物組結(jié)構(gòu)十分復(fù)雜。本課題組前期采用基因芯片及454焦磷酸測(cè)序?qū)Σ煌巳糊x易感兒童的牙菌斑微生物進(jìn)行了系列研究，其結(jié)果進(jìn)一步凸顯了微生物致齲的復(fù)雜性，微生物與齲病的關(guān)系尚需進(jìn)一步深化認(rèn)識(shí)。 當(dāng)前，“核心微生物組”是一個(gè)熱門的研究課題。多數(shù)研究表明口腔中可能存在一個(gè)“核心微生物組”,但尚有學(xué)者持不同觀點(diǎn)。本課題組前期研究支持口腔“核心微生物組”存在的可能性，本實(shí)驗(yàn)擬在前期基礎(chǔ)上，進(jìn)一步為口腔“核心微生物組”是否存在提供一定實(shí)驗(yàn)依據(jù)并探索其可能的微生物組結(jié)構(gòu)。 除了口腔細(xì)菌與齲病息息相關(guān)，唾液蛋白組也與齲病關(guān)系密切。同時(shí)，唾液蛋白組是潛在的診斷和預(yù)防局部和系統(tǒng)性疾病的生物標(biāo)記物來源之一。系統(tǒng)探索唾液蛋白組和口腔微生物組多樣性將有助于對(duì)齲病等口腔局部疾患和系統(tǒng)疾病的認(rèn)識(shí)、預(yù)防和診療。 目的：分析比較高齲組和無齲組兒童牙菌斑（唾液）微生物組結(jié)構(gòu)多樣性以及高齲組和無齲組兒童的唾液蛋白組。第一部分從不同分類學(xué)水平（如門、屬等）比較高齲組和無齲組的微生物組結(jié)構(gòu)組成和差異，并探索“口腔核心微生物組”是否存在。第二部分探索唾液蛋白組與齲病的關(guān)系，并推動(dòng)齲易感性相關(guān)生物標(biāo)記物的研究。 方法：以6-8歲學(xué)齡兒童為研究對(duì)象，使用454焦磷酸測(cè)序分析比較高齲組和無齲組牙菌斑（唾液）微生物組結(jié)構(gòu)和種類（實(shí)驗(yàn)一），采用高壓液相柱色譜—串聯(lián)質(zhì)譜分析及對(duì)比高齲組與無齲組唾液蛋白組（實(shí)驗(yàn)二）。 結(jié)果： 實(shí)驗(yàn)一：（1）本實(shí)驗(yàn)平均每個(gè)樣本檢出超過6000個(gè)OTU，總共得到14076個(gè)OTU，歸于18個(gè)門，，28綱,48目,78科,133個(gè)屬。其中高齲組含9265個(gè)OTU（高齲菌斑6042個(gè)OTU，高齲唾液5406個(gè)OTU），歸屬于13個(gè)門，111種屬；無齲組含10080個(gè)OTU（無齲菌斑6757個(gè)OTU，無齲唾液5661個(gè)OTU），歸屬于17個(gè)門,111種屬。（2）總共有15個(gè)菌屬在高齲菌斑組與無齲菌斑組之間存在有統(tǒng)計(jì)學(xué)意義的差異（p0.05），其中5個(gè)菌屬在高齲菌斑組的檢出高于無齲菌斑組，包括放線菌屬、莫拉克斯氏菌屬、史雷克氏菌屬等；10個(gè)菌屬在無齲菌斑組檢出率高于高齲菌斑組，包括伯杰氏菌屬、二氧化碳嗜纖維菌屬、心桿菌屬、德克斯氏菌屬、顆粒鏈菌屬、嗜血菌屬、金氏菌屬、巨型球菌屬、奈瑟氏菌屬等；（3）總共17個(gè)菌屬在高齲唾液組與無齲唾液組之間存在有統(tǒng)計(jì)學(xué)意義的差異（p0.05），其中11個(gè)菌屬在高齲唾液組的檢出高于無齲唾液組，包括放線桿狀菌屬、丁酸弧菌屬、德克斯氏菌屬、戴阿利斯特桿菌屬、乳桿菌屬、丙酸弧菌屬、假支桿菌屬、鏈桿菌屬等；6個(gè)菌屬在無齲唾液組檢出率高于高齲唾液組，包括卟啉單胞菌屬、嗜血菌屬、不動(dòng)桿菌屬、小鏈菌屬、戴爾福特菌屬等；（4）公認(rèn)致齲菌如變形鏈球菌、內(nèi)氏放線菌、乳桿菌在各樣本中檢出率較低，在高齲組、無齲組之無明顯差異；（5）10個(gè)高齲菌斑樣本與10個(gè)無齲菌斑樣本共有27個(gè)菌屬，包括：放線菌屬、伯杰氏菌屬、二氧化碳嗜纖維菌屬、毛單胞菌屬、棒狀桿菌屬、德克斯氏菌屬、梭桿菌屬、金氏菌屬、孿生菌屬、顆粒鏈菌屬、約翰森氏菌屬、纖毛菌屬、奈瑟氏菌屬、卟啉單胞菌屬、普氏菌屬、鏈球菌屬、密螺旋體屬、韋榮菌屬等；10個(gè)高齲唾液樣本與10個(gè)無齲唾液樣本共有24個(gè)菌屬，包括：放線菌屬、伯杰氏菌屬、彎曲桿菌屬、二氧化碳嗜纖維菌屬、棒狀桿菌屬、德克斯氏菌屬、梭桿菌屬、孿生菌屬、顆粒鏈菌屬、約翰森氏菌屬、纖毛菌屬、奈瑟氏菌屬、消化鏈球菌屬、卟啉單胞菌屬、普氏菌屬、丙酸菌屬、羅氏菌屬、月形單胞菌屬、鏈球菌屬、韋榮菌屬等。 實(shí)驗(yàn)二：（1）本實(shí)驗(yàn)有定量信息的人蛋白質(zhì)1626個(gè)，從高齲組及無齲組唾液樣本中共檢出642個(gè)差異表達(dá)蛋白，包含基質(zhì)金屬蛋白酶9、粘蛋白MUC7、乳鐵蛋白、碳酸酐酶6、天青殺素、冷凝集素等可能與齲病密切相關(guān)的蛋白；（2）本實(shí)驗(yàn)鑒定出的蛋白GO分析提示：蛋白質(zhì)分子功能中，最多的是蛋白結(jié)合性（28.82%），其次是催化活性（22.58%），然后是金屬結(jié)合性（8.74%）及核酸結(jié)合（8.58%）；就參與生物學(xué)過程來說，參與代謝過程的蛋白質(zhì)較多（19.3%），其次為調(diào)節(jié)過程（12.39%）。 結(jié)論：（1）本課題組大部分相關(guān)實(shí)驗(yàn)均發(fā)現(xiàn)韋榮菌屬在高齲菌斑組檢出高于無齲菌斑組，其存在致齲性可能性較高，具體作用有待進(jìn)一步研究求證；（2）菌斑二氧化碳嗜纖維菌屬等與齲病呈負(fù)相關(guān)關(guān)系，唾液二氧化碳嗜纖維菌屬與齲病呈正相關(guān)關(guān)系，其構(gòu)成比可作為潛在的監(jiān)測(cè)齲易感性的指標(biāo)；（3）牙菌斑中多種細(xì)菌參與了齲病過程（如韋榮菌屬、放線菌屬、莫拉克斯氏菌屬、史雷克氏菌屬等），可不需要大量變形鏈球菌、乳桿菌等公認(rèn)致齲菌定植，齲病是多種菌斑微生物共同作用的結(jié)果，支持“菌斑微生態(tài)學(xué)說”；（4）放線菌屬、伯杰氏菌屬、二氧化碳嗜纖維菌屬、梭桿菌屬、孿生菌屬、奈瑟氏菌屬、卟啉單胞菌屬、普氏菌屬、鏈球菌屬、韋榮菌屬等可能是“核心微生物組”的一部分，支持口腔“核心微生物組”的存在；（5）本研究初步表明高齲組兒童唾液蛋白總量及種類均高于無齲組， MMP9、MUC7、LTF、CA6、AZU、冷凝集素等高齲組與無齲組之間的差異表達(dá)蛋白，可能為進(jìn)一步探索齲易感性相關(guān)生物標(biāo)記物提供了一定的實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R788.1

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