Analysis Of The Composition Of Subgingival Bacteria And The Characteristics Of TCR? Chain CDR3 Group In Peripheral Blood Of Patients With Moderate To Severe Periodontitis With Alzheimer's Disease

Objective: Using high throughput sequencing(HTS)to analyze the structure of the subgingival plaque and the characteristics of TCR? chain CDR3 group library in peripheral blood of patients with moderate to severe periodontitis with Alzheimer's disease(AD),Preliminary exploration of the relationship between chronic periodontitis(CP)and AD,to provide a basis for its possible related mechanism research.Methods: According to the inclusion and exclusion criteria,five patients with periodontitis from the periodontal Department of the Affiliated Stomatological Hospital of Zunyi Medical University(group P),eight patients with AD and moderate severe periodontitis from the Department of Neurology of the Affiliated Hospital of Zunyi Medical University and the Department of geriatrics of the second people's Hospital of Guizhou Province were collected(group PAD).Age,sex,number of missing teeth,bleeding on probing(BOP),probing depth(PD),clinical attachment loss(CAL)and mini mental state examination(MMSE)were recorded;The subgingival plaque and peripheral blood of each group were collected and sent to Wuhan Huada gene company for high throughput sequencing.The structural characteristics of subgingival plaque and the characteristics of CDR3 region of TCR? chain in peripheral blood of each group were analyzed by graphpad8,SPSS17.0,and the differences between the two groups and the relationship between the two groups and AD were analyzed.Results:1.There were no significant differences in gender,age,bleeding on probing(BOP),probing depth(PD),and clinical attachment loss(CAL)between the two groups;The number of tooth loss in the PAD group was higher than that in the P group,and thedifference was statistically significant(p <0.05).The score of the MMSE scale in the PAD group was lower than that in the P group,and the difference was statistically significant(p<0.05).2.At the level of phylum,there were higher abundance of Bacteroides,Firmicutes,Fusobacteria,and there was no significant difference between the two groups(P > 0.05);the top four abundance of subgingival plaque in the two groups were the same,which were Porphyromonas,Treponema,Prevotella and fusobacteria,among them,the Prevotella in PAD group was lower than that in P group(P < 0.05),but Pearson correlation analysis showed that there was no significant correlation between the decrease of Prevotella in PAD group and MMSE score(r = 0.187,P = 0.658).At species level,384 species of bacteria were detected in the two groups,and there was no significant difference between the top ten subgingival plaque in the two groups(P > 0.05).3.Comparison analysis of the peripheral blood T cell clone sequences in the two groups showed that the average number of clones in the PAD group was slightly higher than that in the P group,and the difference was not statistically significant(p> 0.05).4.TRBV and J gene frequency disibution and V-J pairing analysis of TCR? chain CDR3 in P and PAD groups of peripheral blood samples.(1)Comparing the expression levels of TRBV genes in groups P and PAD,including a total of 64 TRBV gene fragments,and analyzing the TRBV genes in the top 20 expression levels,the results showed that both groups used TRBV19,TRBV2,TRBV20-1,TRBV29-1 at high frequency;TRBV27 for low frequency;of which TRBV10-3,TRBV12-3,TRBV15,TRBV19,TRBV20-1,TRBV24-1,TRBV27,TRBV5-1,TRBV6-1,TRBV6-5,TRBV7-6 are slightly more frequently expressed in group PAD than in group P,The expression frequency of TRBV10-1,TRBV10-2,TRBV2,TRBV29-1,TRBV7-2,TRBV7-3,TRBV7-8,TRBV7-9,TRBV9 in group P was slightly higher than that in group PAD.After statistical analysis,there was no significant difference between the two groups(p> 0.05).(2)Comparing the expression levels of TRBJ genes in P and PAD groups,the results show that TRBJ1-1,TRBJ1-2,TRBJ1-5,TRBJ2-1,TRBJ2-7 were used at high frequencies;TRBJ1-4,TRBJ2-4,TRBJ2-6 were used at low frequencies.TRBJ1-2,TRBJ1-3,TRBJ1-5,TRBJ2-4,TRBJ2-6 were more frequently expressed in group P than in group PAD.The expression frequency of TRBJ1-1,TRBJ1-4,TRBJ1-6,TRBJ2-1,TRBJ2-2,TRBJ2-3,TRBJ2-5,TRBJ2-7 in group PAD were higher than those in group P.After statistical analysis,there were no statistically significant differences(p> 0.05).(3)Analysis of the V-J gene pairing and retrieval of 13 samples of peripheral blood from group P and PAD.All 13 samples had high-frequency pairing of TRBV20-1-TRBJ2-7.The frequency of TRBV2-TRBJ2-7 in group P was significantly higher than that of Group PAD,The difference was statistically significant(P < 0.05),but Pearson correlation analysis showed that there was no significant correlation between the decrease of TRBV2-TRBJ2-7 frequency and MMSE score in PAD group(r =-0.044,P = 0.917).5.Analysis of the amino acid length of the TCR? chain CDR3 group of T cells in the peripheral blood samples of the two groups of P and PAD,showing a clock-shaped distribution with a midline of 14-15 amino acids in group P and a midline clock with 14 amino acids in group PAD,statistical analysis showed no significant difference in amino acid length between the two groups(p> 0.05).6.No common sequence was found in the top ten of TCR? chain CDR3 repertoires of the two groups of samples P and PAD.A common highly conserved amino acid sequence was found in the two groups,that is,"ETQY,YGYT,NEQF,YEQY".A single conserved amino acid sequence "EKLF" was found in four samples of group PAD,but not in group P,which suggested that the unique conserved amino acid sequences may be related to the unique antigens in PAD group..Conclusion: 1.The composition of subgingival bacteria were different between P and PAD group;2.There was no significant difference in the diversity of TCR ? chain CDR3 group,the use of V and J genes,and the length of amino acids between the two groups of P and PAD.3.There were differences in the selection and use of V-J pairing gene in TCR ? chain CDR3 group library,and highly conserved amino acid sequences in PAD group,suggesting that there might be some differences in the characteristics of TCR ? chain CDR3 grouplibrary between the two groups,and differences were found in the two groups Subgingival plaque,it is speculated that the proportion of subgingival plaque in the two groups and different species may affect the peripheral TCR? chain CDR3 repertoire.