龋源性牙髓炎的可疑致病微生物16S rDNA测序分析

发布时间：2018-06-24 12:58

本文选题：16SrDNA + 牙髓感觉测试　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的通过获取龋源性可复性牙髓炎的龋齿腐质进行16SrDNA检测,探索性研究因龋齿引起牙髓炎的细菌种类和数量特征与不同牙髓炎症状态的相关性,以期是否能找到一种简便快捷的牙髓状况判定方法。对象和方法取自天津医科大学口腔医院牙体牙髓科的可复性牙髓炎病例,通过牙髓活力问卷调查表,获取牙髓活力判断的临床检查指标,按照美国牙髓病学协会(American Association of Endodontists,AAE)的诊断标准,明确诊断。共收集35例病例,其中龋源性可复性牙髓炎22例、龋源性不可复性牙髓炎8例和无症状深龋5例。再进一步细分为4组,分别是无热刺激痛的可复性牙髓炎组(A1组)15例,有热刺激痛的可复性牙髓炎组(A2组)7例,有症状不可复性牙髓炎组(B4组)8例,正常牙髓深龋对照组(B3组)5例。上橡皮障,收集龋齿窝洞腐质样本,送检。采用MiSeq测序仪,对4组共35例样本的龋齿腐质内细菌进行16S rDNA高变区测序,首先进行可操作分类单元(Operational Taxonomic Units,OTUs)聚类分析,研究具体OTUs及细菌的菌体种类,然后进行6种α多样性分析,分析菌群物种的丰度,这6种指数分别为丰富度稀疏指数(Observed-species),香农-威纳指数(Shannon-Wiener Index),辛普森菌群多样性指数(Simpson’s diversity Index),Chao1菌种丰富度指数,ACE指数,测序深度指数(Good-coverage)。然后进行β多样性分析,研究4组组间和35例样本间菌群分布的差异性,包括3个指标:主坐标分析(Principal Co-ordinates Analysis,PCoA分析)、非度量多维尺度分析(non-metric multi-dimensional scaling,NMDS)和LEfSe分析(Linear Discriminant Analysis Effect Size)。然后,将样本的OTUs检测分析数据与已知明确的致龋菌、牙髓病细菌种属进行比对,即首先将样本测序数据与临床检查指标数据行去趋势分析(Detrended Correspondence Analysis,DCA分析),得到4个序列长轴的梯度值。参考第一序列长轴的梯度范围,最大值为2.52,小于3,因此进一步做冗余分析(Redundancy Analysis,RDA分析)。结果35例患牙的龋齿腐质分析,得到OTUs聚类6881种,共同优势菌属为乳杆菌属、放线菌属、拟杆菌属等。α多样性分析见B4组分别与B3、A1、A2组比较无统计学差异(P0.05)。β多样性分析可见与B3组比较,各组分别有多种细菌的含量较高,有统计学差异(P0.05)。A1组主要为放线菌和乳杆菌,A2组主要为β变形菌,B4组为加氏乳杆菌(L.gasseri)、发酵乳杆菌(L.fermentum)、干酪乳杆菌(L.casei)。链球菌属(Streptococcus)、奈瑟氏菌属(Neisseria)细菌数量与冷刺激痛强度、热刺激痛强度呈正相关关系;放线菌属(Actinomyces)细菌含量与热刺激痛强度呈正相关关系;嗜冷杆菌属(Psycnnlyobacter)、假单胞菌属(Pseudomonas)、气单胞菌属(Aeromonas)、拟杆菌属(Bacteroides)的含量与牙髓电活力测试数值呈正相关关系;乳杆菌属细菌的数量与冷刺激痛时间(CST)、热刺激痛时间(HST)、自发痛强度(Sp)及患牙、对照牙间电活力数值差(CT)呈正相关关系。结论1.从正常牙髓深龋到无热刺激痛的可复性牙髓炎,有热刺激痛的可复性牙髓炎,再到有症状不可复性牙髓炎,这一动态病变进展过程中微生物多样性具有一定的差异,细菌数量和丰度不同;正常牙髓、可复性牙髓炎的细菌菌群多样性差别不大,而不可复性牙髓炎的细菌菌群多样性降低。无热刺激痛的可复性牙髓炎菌群分布与正常牙髓深龋类似,而同时有冷热刺激痛的可复性牙髓炎菌群分布与有症状不可复性牙髓炎相似。2.乳杆菌属与自发痛及冷热刺激痛持续时间长短相关,链球菌属与冷热刺激痛强度相关;奈瑟菌属、韦荣氏菌属、弓形杆菌属、嗜冷菌属、假单胞菌属、气单胞菌属、拟杆菌属等与牙髓电活力测试数值正相关。乳杆菌属可能与深龋逐渐发展为不可复性牙髓炎密切相关。链球菌属、奈瑟菌属、韦荣氏菌属、弓形杆菌属、嗜冷菌属、假单胞菌属、气单胞菌属、拟杆菌属与牙髓的炎症继续发展从可复性牙髓炎转变为不可复性牙髓炎有一定关系,而放线菌可能与牙髓炎与这一病变进展过程无直接关系。
[Abstract]:In order to find a simple and convenient method to determine the bacterial species and quantity of dental pulpitis , there were 5 cases with chronic dental pulpitis ( group A1 ) , 8 cases with chronic dental pulpitis and 5 cases of asymptomatic deep caries . The results showed that there were 22 cases of pulpitis , 8 cases with chronic dental pulpitis and 5 cases of asymptomatic deep caries . NMDS)鍜孡EfSe鍒嗘瀽(Linear Discriminant Analysis Effect Size). Then , comparing the OTUs detection and analysis data of the samples with known definite cariogenic bacteria and dental pulp bacteria species , the sample sequencing data and the clinical examination index data line de - trend analysis ( DCA analysis ) were first carried out to obtain the gradient values of the long axis of the four sequences . The gradient range of the long axis of the first sequence was referenced , the maximum value was 2.52 , and the maximum value was 2.52 , which was less than 3 , thus further redundant analysis ( RDA analysis ) was further done . Results The results showed that there were no statistically significant differences in the quantity of bacteria and the intensity of cold - stimulating pain ( P0.05 ) . The results showed that there were no statistically significant differences in the quantity of bacteria and the intensity of cold - stimulating pain ( P0.05 ) . The inflammation of the genus Aeromonas , the pseudobacterium and the pulp continued to develop from the allergic pulpitis to the irreversible pulpitis , and the actinomyces may have no direct relationship with the progression of the lesion .
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.31

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