| Objective:Using high-throughput sequencing（HTS）technology and 16S r DNA sequencing to analyze the composition characteristics of subgingival microflora of SD rats in periodontal health state and moderate to severe periodontitis state,etc.To explore the scientificity and rationality of promoting the formation of experimental periodontitis in rats by oral inoculation of pathogenic bacteria,and to provide the basis and evidence for the establishment of experimental periodontitis model in rats in the future.Methods:1.Twenty 9-week-old SD rats were fed for one week and randomly divided into two groups:controled(group N.SD)（n=10）and severe periodontitis(group P.SD)（n=10）.Simply ligate the first and second molars in maxillary of rats to build the heavy experimental periodontitis model group,and build normal model group with the same food and water.After 10 weeks,stereomicroscope was used to observe the alveolar bone absorption,and identify the formation of severe periodontitis.Subgingival plaque samples were collected respectively,and stored in the-80℃refrigerator.2.Subgingival plaque samples from patients treated in the Affiliated Stomatological Hospital of Zunyi Medical University were collected,including 10 healthy periodontitis patients（group N.H）and 10 moderate or severe periodontitis patients（group P.H）.The basic information and periodontal clinical indicators（bleeding index,periodontal pocket depth,the number of tooth loss,etc.）were recorded respectively.Subgingival plaque was collected and stored in-80℃refrigerator.3.All subgingival plaque samples were sent to Novegene.After the quality inspection,DNA of each group was extractedand and amplify the 16S r DNA V3V4 region by PCR,and sequence by Illumina Nova to obtain the original data and analyze the structuralcharacteristics and diversity of microbial community in rat and human subgingival plaque.Results:1.Oral microorganisms were classified into 68 phyla,153 classes,327 orders,472 families,767 genera and 490 species in the 33 samples by OTU annotation.2.The common microflora of the four groups was low.The species composition and abundance between group N.SDand P.SDwere more similar,the same between group N.Hand P.H,and the species composition and abundance were little similar between the former two and the latter two.3.The diversity of group N.SD,group P.SD,group N.H,and group P.His increasing.The diversity of community composition in group P.SDand group P.Hwas significantly different（p<0.01）,and the diversity of community composition among group N.SD,group P.SD,group N.H,and group P.Hwas statistically significant difference（p<0.05）,indicating that there was no significant similarity among the four groups.Group P.SDand group P.Hwere statistically significant difference in the contents of major periodontal pathogen,the same with dominant bacteria in P.SDgroup（p<0.01）.4.The common dominant phyla of group N.SDand group P.SDwere Proteobacteria,Firmicutes,Bacteroidetes,Fusobacteria,Actinobacteria,Desulfurobacteria.The common dominant bacteria genera were:Rodentibacter,Bacteroidetes,Succinivibrio,Desulfurization,Escherichia-Shigella;The common dominant strains were:Pasteurellaceae＿bacterium,Bacteroides＿vulgatus,Bacteroides＿xylanisolvens,Escherichia＿coli.The dominant strains in group N.SDwere P.bacterium,B.vulgatus,M.Muris,Faecalibacterium＿prausnitzii,B.xylanisolvens,E.coli;The dominant strains in P.SDgroup were D.sp＿G11,B.xylanisolvens,P.bacterium,Bacteroides＿sartorii,Neisseria＿sp＿10022,B.vulgatus,Lactobacillus＿murinus,Bacteroides＿uniformis,E.coli,Veillonella＿ratti,Morganella＿morganii,Lachnoclostridium＿sp＿YL32,Fusobacterium＿ulcerans.It was suggested that D.sp＿G11,B.xylanisolvens,N.sp＿10022,L.murinus,B.uniformis,V.ratti,L.sp＿YL32,F.ulcerans were associated with periodontitis in rat,while there was no statistical significance.As the core strains,P.bacterium,B.vulgatus,B.xylanisolvens,E.coli were important members of subgingival microorganism of rat periodontitis.5.LEf Se cluster tree analysis at species level showed the biomakers,that there were 3 species（P.bacterium,B.vulgatus,M.muris）in group N.SD,Haemophilus＿parainfluenzae in group N.H,8 species（D.sp＿G11,B.xylanisolvens,B.Sartorii,N.sp＿10022,B.uniformis,L.murinus,E.coli,V.ratti）in group P.SD,4 species（P.g、P.e、T.d、Neisseria＿mucosa）in group P.H,which were included in the advantages of each species.The classical pathogen of human periodontitis did not play an obvious role in the development of experimental periodontitis in rat with simple ligation.Conclusion:1.There were significant differences in the composition of subgingival plaque flora above human with periodontal health,human with periodontitis,SD rats with periodontal health and SD rats with periodontitis.The abundance of subgingival plaque flora in human and SD rats with periodontitis was higher than that in the healthy periodontal health group;2.The content of classical main pathogenic bacteria of human periodontitis was little in the subgingival plaque of experimental periodontitis in SD rats caused by simple ligation,and was not the dominant bacteria of SD rats;3.Rodentibacter,Bacteroidetes,Succinivibrio,Desulfurization,Escherichia-Shigella,Streptococcus,P.bacterium,B.vulgatus,B.xylanisolvens,E.coli were the core of the subgingival microflora of rats.Streptococcus,Veillonella,Lactobacillus,E.coli,M.morganii,D.sp＿G11,B.xylanisolvens,N.sp＿10022,L.murinus,B.uniformis,V.ratti,L.sp＿YL32,F.ulcerans were associated with the periodontitis in rat. |
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