| Objective: The characteristics of TCR? chain CDR3 group was analyzed by highthroughput sequencing technology in subgingival plaque microbial communities and local gingival tissues of severe chronic periodontitis.The effects of microbial community composition of subgingival plaque on TCR? chain CDR3 group in local gingival tissues was preliminarily explored.The new research ideas and references are provided in order to further study the role of T cells in the pathogenesis of periodontitis.Methods: 1.5 cases of periodontal health(group H)and severe chronic periodontitis(group P)was respectively collected.The subgingival plaque and gingival tissue were collected at one of the sites of tooth extraction before operation,and the gingival tissue was frozen by liquid nitrogen.2.The DNA of each gingival tissue sample was extracted separately.3.Genomic DNA samples and subgingival plaque samples were sent to BGI for library construction and sequencing.4.The raw data were converted and then uploaded to the IMGT / High V-Quest system,and the composition of CDR3 library sequences of each sample TCR? chain was analyzed the pairings of TRBV,TRBJ,V-J in the CDR3 library of TCR? chain in periodontal health and severe chronic periodontitis were analyzed by EXCEL,Graph Pad Prism 6 and Draw Venn Diagram the diversity of CDR3 library,amino acid length,amino acid uptake and nucleotide deletion / insertion,etc..5.The effects of subgingival microbial composition on the CDR3 pool of TCR? chain in gingival tissue was initially explored.Results:1.Microbial genome sequencing in subgingival plaque of patients in group H and group P was analyzed by OUT Venn diagram.The results showed that the number of core species co-colonized in the two groups was relatively large.The analysis of species diversity showed that the OUT Rank curve of group P was narrower than that of group H,and the decline trend was steep,suggesting that the species richness and uniformity of subgingival flora in group P was lower than that in group H.2.Statistical analysis of the subgingival plaques in the H and P groups at the phylum level showed that the number of the Actinomycetes and Proteobacteria of group H was significantly higher than that of group P(P<0.05),while the number of the Tenericutes of group P was significantly higher than that of group H(P<0.05),and the number of the Bacteroidetes,Spirochaetes and Synergistetes of group P was significantly higher than that of group H(P<0.05).At the genus level,the number of Bulleidia,Butyrivibrio,Filifactor,and Tannerella of group P was significantly higher than that of group H(P<0.05).At the species level,the number of Acinetobacter guillouiae,Capnocytophaga ochracea,Rothia aeria,and Streptococcus infantis of group H was significantly higher than that of group P(P<0.05),while the number of Porphyromonas endodontalis,Pi of group P was significantly higher than that of group H(P < 0.05).3.The ratios of functional unique sequences to functional total sequences of the TCR?chain CDR3 library in each gingival tissue sample were: H1:12.15%,H2: 12.49%,H3:11.85%,H4: 11.39%,H4: 11.94%,P1: 11.46%,P2: 11.39%,P3: 8.43%,P4: 8.14%,P5:11.57%.The ratio of unique effective sequences in group H was higher than that in group P,suggesting that there were more abundant T cell clones in the healthy periodontal tissues.4.Clonal amplification of TCR? chain CDR3 in each gingival tissue sample: The inverse Simpson's diversity index(1/DS)of each sample was: H1: 313.12,H2: 91.06,H3: 167.77,H4: 140.44,H5:514.62,P1:304.54,P2:492.33,P3:199.39,P4:79.37,P5:73.97.Statistical analysis showed that 1/DS in group H was higher than group P,but the difference was not statistically significant(P>0.05).The identical sequences were not found in the top 5high-frequency clonal proliferation sequences of each sample,but the amino acid motif "YEQY" was found to be high-frequency in both groups;and the amino acid motif "ETQY" was only found in group P.The unique and highly conserved amino acid motifs in group P may be related to the immune response of periodontal local environment to specific antigen in patients with severe chronic periodontitis.5.The TRBV,TRBJ gene family and V-J pairings of the TCR? chain CDR3 group library were extracted from each gingival tissue sample:(1)The selected TRBV genes of high frequency(>2%)in both group H and group P:TRBV 10-3,TRBV19,TRBV2,TRBV 20-1,TRBV 29-1,TRBV 5-1,TRBV 7-2,TRBV 7-9and TRBV9.According to statistical analysis,the uptaken frequency of TRBV 6-8 gene by TCR? chain CDR3 library of total T cells in gingival tissue in group P was significantly higher than that in group H(P < 0.05).(2)The high frequency(> 5%)of TRBJ genes in both H group and P group included:TRBJ 1-2,TRBJ 1-5,TRBJ 2-1 and TRBJ 2-7.Statistical analysis showed that compared with group H,the frequency of TRBJ 2-5 gene uptake by the TCR? chain CDR3 library of total T cells in the gingival tissue in group P was significantly increased(P < 0.05).(3)The common dominant pairation(> 1%)genes of H and P included: TRBV19-TRBJ2-7,TRBV2-TRBJ2-7,TRBV20-1-TRBJ1-2,TRBV20-1-TRBJ2-1,TRBV5-1-TRBJ2-7,TRBV7-9-TRBJ2-7.According to the statistical analysis,compared with group H,the frequency of TRBV10-3-TRBJ2-5,TRBV11-2-TRBJ2-3,TRBV12-5-TRBJ1-5,TRBV29-1-TRBJ2-5,TRBV30-TRBJ2-1,TRBV5-1-TRBJ2-5,TRBV5-6-TRBJ2-7,TRBV5-8-TRBJ2-1,TRBV6-8-TRBJ1-2,TRBV7-9-TRBJ2-5,TRBV7-9-TRBJ2-5,TRBV9-TRBJ2-1,gene pairing and gene selection was significantly higher in group P than in group H(P < 0.05).The genetic diversity may be related to the immune response to different antigens in the periodontal environment.6.Extraction and length distribution of TCR CDR3 AA in each gingival tissue sample: The amino acid length distribution of TCR? chain CDR3 in the gingival tissues of group H and group P was normal with the length of 12 amino acids as the main part.Through statistical analysis,there was no significant difference in amino acid length distribution between the two groups(P > 0.05).Serine(S)was taken from gingival tissue of both group H and group P at high frequency.Through statistical analysis,there was no significant difference in amino acid uptake between the two groups(P > 0.05).The analysis on amino acid site showed that alanine(A)was taken with high frequency at the 105 th site in both group H and group P,and serine(S)was taken with high frequency at the 106 th and 107 th site,and glutamic acid(E)was taken with high frequency at the 115 th site,and glutamine(Q)was taken with high frequency at the 116 th site,and tyrosine(Y)was taken with high frequency at the 117 th site;while the diversity of the uptaken amino acid at 108 th,109th,110 th,112th,113 th,114th site was presented,affecting the clonal proliferation of the TCR? chain CDR3 library.7.The overlapping sequences of unique amino acid in the TCR? chain CDR3 library of each gingival tissue sample: the number of unique amino acid sequences shared by the TCR? chain CDR3 library of 5 patients in group H was 129,and the number of that in group P was 11;the number of unique amino acid sequences shared by 10 samples of both group H and group P was 6.8.The deletion and insertion of nucleotides in CDR3 library of TCR? chain in both group H and group P: the cutting and insertion of nucleotides in CDR3 region of TCR chain in H and P groups were statistically analyzed the number of V deletions,N1 insertions,D5 deletions in the CDR3 region of TCR? chain in gingival tissue of P group were significantly decreased(P< 0.05).It is suggested that the composition of CDR3 receptor library of TCR? chain in H group is more abundant,which may induce immune response to more antigens.Conclusion:1.The subgingival flora composition of patients in group H and group P was different.The diversity of subgingival flora in group P was lower than that in group H.The patients in both two groups had their own unique dominant flora,and the differences of composition of species was significant.2.The diversity of CDR3 library of TCR? chain in H group was higher than that in P group,but there were no significant differences in amino acid length and uptaken amino acid.Some TRBV,TRBJ gene families and TRBV-TRBJ pairs were significantly different,and the number of deleted / inserted nucleotides in the CDR3 region was also significantly different.It is suggested that the subgingival microbial composition of group H and group P might affect the CDR3 library of TCR? chain in gingival tissue,and the differential gene expression might be related with the specific immune response caused by different antigens in the periodontal microenvironment. |
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