| Objective: High-throughput sequencing(HTS)technology was used to analyze the characteristics of the subgingival microbial community under different periodontal conditions,the heterogeneity of the gingival / tumor tissue TCR ? chain CDR3 repertoire,and its association with oral squamous cells(OSCC),with a view to providing new ideas and basis for the occurrence mechanism and prevention of OSCC.Methods: According to the diagnosis and inclusion criteria,a total of 15 related cases were collected as study subjects,and the study subjects were divided into three groups: 5 patients with moderate to severe chronic periodontitis(CP)with OSCC(gingival site),5 patients with moderate to severe CP patients,and 5 patients with periodontal health were named: T,C,H groups;the bacteria can be divided into subgingival plaque group(bp group)and gingival tissue group / OSCC tumor tissue group(g group);record the basic information(gender,age)of the study subjects and clinical periodontal indicators(probing depth,periodontal attachment level,probe bleeding);corresponding to the tooth position or extraction tooth position in the surgical area of 15 subjects,take subgingival plaque samples and collect two copies of local periodontal tissue(gingival tissue / gingival squamous cell carcinoma tissue)corresponding to the tooth position,and store them at-80 ? in refrigerator;send each sample to Huada Gene Technology(Wuhan,China),after passing the quality inspection,DNA was extracted from three groups of subgingival plaque(bp group)and gingival / tumor tissue(g group),16 S r DNA V4 region was amplified by PCR,and Hi Seq2500 was used for sequencing;Three groups of gingival / tumor tissues were used for sequencing the immunohistochemical library to extract RNA,and the Hiseq4000 platform was used for sequencing the immune repertoire.The sequencing data were obtained from Wuhan Huada Gene Corporation,and the IMGT / High V-QUEST platform(www.imgt.org),QIIME,Mothur,VDJtools and other software were used to analyze the subgingival plaque and local periodontal tissue microbial community structure characteristics Diversity,analysis of the composition of the TCR? chain CDR3 repertoire in each sample,the acquisition and pairing of the TRBV and TRBJ gene families,the diversity of the CDR3 repertoire(chao1 index,chao E index,Shannon index),etc.Results:1.Analysis of microbial OTU venn diagrams of gingival plaque and gingival / tumor tissue in three groups of patients found that a large number of core species were co-colonized in groups H,C,and T.The analysis of the diversity of subgingival plaque species showed that the diversity of the subgingival flora in the C-bp group was higher than that in the H-bp group(P <0.05),and the diversity of the subgingival flora in the T-bp group was lower than H-bp group(P <0.05);analysis of species diversity in gingival / tumor tissue showed that the microbial diversity was highest in the gingival tissue of the H-g group,followed by the C-g group,and the lowest in the T-g group,but the difference was not statistically significant(P> 0.05);In addition,the microbial diversity in the gingival / tumor tissues of the H,C,and T groups was slightly higher than that of the subgingival plaques,and there was no significant difference(P> 0.05).2.At the phylum level,a statistical analysis of the relative abundance of sub-gingival plaques and gingival / tumor tissues in the three groups of patients showed that: the fusobacteria in the C-bp group was higher than the T-bp group(P < 0.05),the proteobacteria in the T-bp group were higher than those in the C-bp group(P <0.01),the spirochaetes in the C-bp group was significantly larger than the T-bp group(P<0.05);The relative abundance of C-bp group,H-bp group,T-bp group fusobacteria and spirochaetes was statistically significant;the fusobacteria in the H-g group was significantly larger than that in the C-g and T-g groups(P <0.05),and the proteobacteria in the T-g group was higher than that in the H-g group(P <0.05),The decrease in the relative abundance of proteobacteria in the H-g group,T-g group and C-g group was statistically significant;the differences in the phylum level between the gingival / tumor tissues and subgingival plaques in the H,C,and T groups: H-bp group proteobacteria was higher than the H-g group(P <0.01),C-bp group fusobacteria was higher than the C-g group(P <0.01).There was no significant difference between each phylum of the T-bp group and the T-g group.3.At the genus level,the unique dominant bacteria in the T group are: pseudomonas,peptostreptococcus,and granulicatella,of which T-bp group,peptostreptococcus and granulicatella significantly larger than the C-bp group(P <0.05);the unique dominant bacteria in C group: filifactor,filifactor of the C-bp group is significantly larger than the T-bp group(P <0.05);the H group is unique The dominant bacteria: lactobacillus and megasphaera,but there is no significant difference.The differences of relative abundance levels among the dominant genus groups in the H,C,and T groups were compared: streptococcus in the T-bp group was significantly larger than that in the C-bp group(P <0.05),and treponema in the C-bp group was significantly larger than the T-bp group(P <0.05),prevotella in the C-bp group was significantly larger than that in the H-bp group(P <0.05);capnocytophaga in the T-g group was significantly larger than in the H-g group(P <0.05);dialister in the H-g group was significantly larger than the C-g group(P <0.05);the differences in genus levels of gingival / tumor tissues and subgingival plaques in the H,C,and T groups showed that: the C-bp group of fusobacterium(P <0.01)and prevotella(P <0.05)was significantly larger than the C-g group,dialister in the H-bp group was significantly smaller than the H-g group(P<0.05),and neisseria was significantly larger in the H-bp group than in the H-g group(P <0.05),There was no significant difference between T-bp group and T-g group.4.At the species level,comparison of species relative abundance levels between groups:streptococcus infantis was significantly higher in the T-bp group than in the C-bp group(P <0.05),veillonella parvula in the T-g group was significantly smaller than in the H-g group(P <0.05).The reduction of veillonella parvula in the group T-g,C-g and H-g was statistically significant;treponema socranskii and prevotella melaninogenica in the C-bp group were significantly larger than in the H-bp group(P<0.05);prevotella nigrescens was significantly larger in the H-g group than in the T-g group(P <0.05).5.Redundant analysis of microbial species and environmental factors(PD,AL,BOP)in gingival / tumor tissue samples showed that: The included angle between pseudomonas,granulicatella,peptostreptococcus and each environmental factor is an acute angle,showing a positive correlation;the included angle between prevotella nigrescens,veillonella parvula and each environmental factor is an obtuse angle,showing a negative correlation.6.Diversity analysis of TCR ?-chain CDR3 repertoire in three groups of gingival /tumor tissue samples: TCR ?-chain CDR3 repertoire diversity in group C patients was higher than that in group H,and group T had the lowest diversity,but there was no statistical difference;three groups both highly conserved amino acid(Amino Acids,AA)motifs "YEQY" and "ETQY" are present,and appear most frequently in group H,which may be related to the stimulation of common epitopes in the environment;unique conservation was found in group T AA motif "NEQF",this motif was not found in groups H and C.7.Analysis of TCR ? chain CDR3 repertoire TRBV,TRBJ gene family and V-J pairing in gingival tissue samples of H,C and T groups:(1)The TRBV gene families taken by the three high-frequency groups H,C,and T are TRBV2 and TRBV20-1,with significant differences(P <0.05);The frequency of accessing TRBV5-1 and TRBV2 gene families in group T was significantly higher than that in group H(P<0.05),and the frequency of accessing TRBV2 gene family in group T was significantly higher than that in group C(P<0.05).(2)The TRBJ gene families commonly used in the H,C,and T groups are TRBJ2-7,TRBJ2-5,TRBJ1-1,TRBJ2-2,and TRBJ1-2,but there is no significant difference(P>0.05);(3)H,C,T three common dominant pairs(> 1%)genes include: TRBV20-1-TRBJ2-7,TRBV20-1-TRBJ2-5,TRBV2-TRBJ2-7,TRBV29-1-TRBJ1-1,TRBV28-TRBJ2-7.The differential access of genes may be related to the immune response produced by the local periodontal environment to different antigens.8.Acquisition and length analysis of TCR ? chain CDR3 AA in three groups of gingival / tumor tissue samples: Groups H and C were distributed in a bell shape,mainly with 13-15 AA lengths,and group T were mainly with 14-15 AA lengths Most were used.9.Unique AA overlapping sequences in the TCR ?-chain CDR3 repertoire of the three groups of gingival / tumor tissue samples: No overlapping AA sequences were seen inthe samples of group H and C,and there were 5 overlapping AA sequences in the samples of group T.Conclusion:1.The composition of the subgingival flora of the three groups H,C,and T is different.Each of the three groups has its own unique dominant flora,and the composition of the species is significantly different.2.The increase of peptostreptococcus and granulicatella in subgingival plaques of patients with moderate to severe CP may be related to the occurrence and development of OSCC.3.TCR ?-chain CDR3 repertoire in gingival / tumor tissue of patients in group H,C,and T group.TCR ?-chain CDR3 repertoire diversity in group T patients was lower than that in group H,while the diversity in group C was higher than that in group H,but The diversity is not significant.Significant changes in the acquisition of TRBV2 genes,partial TRBJ genes,and paired access of TRBV-TRBJ genes,suggesting that the microbial composition of subgingival plaque may affect the TCR ?-chain CDR3 repertoire in gum The differential expression may be related to the immune response induced by specific antigens recognized in different periodontal microenvironments.Among gingival / tumor tissue TCR ?-chain CDR3 repertoires in groups H,C,and T,the diversity of CDR3 repertoires in group T was lower than those in groups C and H,but the differences were not significant.Significant changes in the acquisition of TRBV5-1 and TRBV2 genes,the retrieval of some TRBJ genes,and the matching and retrieval of TRBV-TRBJ genes indicate that the microbial composition of subgingival plaques may affect the TCR ?-chain CDR3 repertoires in gingival / tumor tissue,this may be related to the immune response of different periodontal microenvironments. |
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