Study On The Characteristics Of Immune Repertoire In Vascular Diseases Caused By Disorders Of Lipid Metabolism

BackgroundThe prevalence of dyslipidemia in China shows a trend of rapid increase,lipid metabolism disorders are closely related to the pathogenesis of diabetes,hypertension,non-alcoholic fatty liver and atherosclerosis.Disorders of lipid metabolism can cause hyperlipidemia and lipotoxicity,further causing abnormal immune inflammation,which in turn will further promote lipid metabolism disorders.Atherosclerosis(AS)is a major vascular complication of dyslipidemia.AS is a disease that caused by the deposition of lipid in the vessel,it will lead to endothelium injury,both inflammatory cells and inflammatory cytokines are accumulated,and ultimately,it will develop into lipid stripes or plaques,resulting the luminal morphology lesions.AS is a typical representative of the mutual promotion of lipid metabolism disorders and immunoinflammatory and the key factor in its pathogenesis is dyslipidemia.T lymphocytes and B lymphocytes are the main components of immune response,which mediate cellular immunity and humoral immunity respectively.They play an important role in immunoinflammatory and play a role as executor and regulator.T lymphocytes and B lymphocytes play an important role in the immunoinflammatory caused by lipid metabolism disorders and are closely related to the occurrence and development of AS.Although a large number of clinical and basic studies have demonstrated that different T cell subsets exist in different AS stage that can influence disease progression and different B cell subsets have different effects on AS.However,the specific mechanisms by which T cells and B cells play a role in AS are still not completely elucidated.In particular,how T cell immune responses activate and regulate this precise mechanism in AS and how B cells affect AS,Whether B-2 B cells affect AS by secreting Immunoglobulin G(IgG)still remain poorly understood.The more T cells and B-cell subsets involved in this initiation and regulation process,the more complex the mechanisms involved.The reason for this complex phenomenon may be the diversity of T cell subsets and B cell subsets as well as the variability of T cell receptor(TCR)and B cell receptor(BCR).Therefore,the study of T cell and B cell immunity in atherosclerotic plaques can further reveal the pathogenesis of AS.Immune repertoire(IR)refers to the sum of B cells and T cells with functional diversity in the circulatory system of one individual at any given time.The diversity of T cell immune repertoire and B cell immune repertoire will decrease and a single,oligo or multiple clones will increase when a specific immune response occurs.Immune Repertoire sequencing(IR-SEQ),which is a high-throughput sequencing method,provides the possibility of studying the diversity and variability of immune repertoire.Next Generation Sequencing provides a high sensitivity,specificity and accuracy which gives a higher reliability for the study of IR.This study will help us to deeply understand the real role of T cell and B cell in AS and also provide more evidence to reveal the mechanism of inflammatory immune abnormalities in AS.ObjectivesWe studied the difference between atherosclerotic plaques and normal subjects peripheral blood in the T cell immune repertoire and B cell immune repertoire,including clone frequency,amino acid length of complementarity determinant region 3(complementarity determining region 3,CDR3),the distribution characteristics of common clones and the utilization of VDJ gene.In order to reveal the composition and changes of cell clones in AS patients at the DNA base level.This will provide more detailed evidence for the pathogenesis of AS.Methods1 56 samples of peripheral blood(5 mL each)from healthy subjects were obtained.Inclusion criteria:clinical manifestations of non-typical angina and ECG changes in the normal subjects,Age>40 years old.Peripheral blood mononuclear cells(PBMCs)were extracted by density gradient centrifugation method.4 AS plaque tissue samples were taken from autopsy specimens and plaque specimens with AS characteristics.2 The TIANamp Genomic DNA Kit was purchased from TIANGEN Biotech(Beijing)Co.Ltd.The genomic DNA of peripheral blood mononuclear cells(PBMCs)and tissues were extracted by spin column method.After each group of gDNA extraction,the same amount of gDNA mixed together to form a normal group of peripheral blood sample pool and AS plaque group sample pool.3 Optimum DNA usage based on DNA concentration and quality.Multiplex PCR(QIAGEN multiplex PCR Kit)was used to capture the CDR3 region by adding the forward and reverse primers.The QIAquick PCR Purification Kit was used to purify the PCR product,and then the library was constructed after the terminal repair and adaptors link.Finally,the constructed library was evaluated by detecting the range of insert and concentration quantification.4 Calculate the amount of data on the machine.The diluted RNA library was added to the Flowcell for bridging PCR amplification and sequencing.5 The filtered raw data sequences were aligned by miXCR,which can analysis different CDR3 sequences and its expression in formation.Besides,it can also identify the amino acid sequence of the CDR3 region and the corresponding clone reading,as well as the number and frequency of VDJ genes.In addition,frequency interval distribution,cumulative frequency distribution and VDJ gene utilization were analyzed by writing scripts and Microsoft Excel 2010 software.6 The data obtained from the experiment were statistically analyzed by Microsoft Excel 2010 and GraphPad Prism 5.Mean ± SEM was used and two-tailed t-test was used for the data statistics comparison.P<0.05 was considered statistically significant.Results1 The comparison of T cell clones frequencyFor the frequency of first 100 clones,the frequency of the AS plaque group was significantly higher than the control subject.In the relatively low frequency range,the number of specific clones of the control group which within the specified interval was significantly higher than the AS plaque group.In the high frequency range(0.006-0.04),the number of specific clones in the AS plaque group was significantly higher than that in healthy subjects.The cumulative frequency has similar results.2 The analysis of T cell clone lengthsFor healthy subjects and the AS plaque group,there was no significant difference for the length distribution of amino acid in T cell clones,and the overall distribution tends was consistent.The amino acid length of most CDR3 sequences is 14 and 15,accounting for 50%.3 The analysis of T cell common clonal proportion distributionCompared to those in the peripheral blood of healthy control subjects,the proportion of common clones(clones are present in more than two subjects)in the atherosclerotic plaques was significantly elevated.The average ratio in AS plaques was 0.38,whereas the average ratio in healthy subjects was about 0.23.The analysis of the common clones of the three subjects showed that the proportion of common clones in the specific clones was significantly higher in the plaques group than in the healthy subjects,and the proportion of the common clones in all clones also significantly increased in the plaques group.4 The analysis of V and J gene utilizationThe utilization of some V genes and J genes in the T cell repertoire of atherosclerotic plaques was significantly higher than that in the healthy subjects,such as TRBV20-1,TRBJ2-5,TRBJ2-1,which suggests that there may be common antigen or pathological stimuli.5 The comparison of B cell clones frequencyFor the frequency of first 100 B cell clones,the frequency of the AS plaque group was significantly higher than the control subject.The number of B cell clones in AS plaques in>0.1%frequency range was significantly higher than that of healthy subjects6 The analysis of B cell clone lengthsThe frequencies of B cell clones for some of the amino acid lengths such as(15,16,17,18,and 20)in the AS plaques group,were significantly higher than those in the healthy subject group.7 The analysis of VJ gene utilization of the B cell repertoireCompared to the healthy subjects,the V-J gene utilization(V-J genotype)of the B cell repertoire in the AS plaques was significantly decreased,but the high-frequency B cell clones was significantly increased,such as V3-21-J4 and V3-23-J4.The utilization rates of certain V genes and J genes in plaques were significantly different from those in healthy subjects(such as IGHV3-21,IGHV3-23,IGHV3-33,IGHJ4).Conclusion1 Compared to those in the peripheral blood of healthy control subjects,the number of high-frequency clones of T cell and B cells in the plaques of atherosclerotic patients were increased.2 Compared to those in the peripheral blood of healthy control subjects,the proportion of T cell common clones in the plaques of atherosclerotic patlents was increased.3 Compared to those in the peripheral blood of healthy control subjects,the utilization rates of V genes and J genes which encoding certain specific T clones and B clones were elevated in the plaques of AS.