High Throughput Sequencing Of The T Cell Receptor CDR3 Repertoire In Patients With Acute Myocardial Infarction

Objective:To sequence and establish the immune repertoires of peripheral blood TCR CDR3 in patients with acute myocardial infarction.Comparing the difference between the acute myocardial infarction group and healthy control group to analyze characteristics of TCR CDR3 repertoires in acute myocardial infarction.Methods:(1)According to the criteria for inclusion and exclusion,people can be divided into the AMI patients group and the healthy control group,and then collect clinical data on gender,age,white blood cell count(WBC),neutrophil percentage(NEUT%),lymphocyte ratio(LY%),monocyte ratio(MONO%),high-sensitivity troponin I(hs-Tn I),the number of lesion vessels for preliminary statistical analysis.(2)The c DNA solution of the total peripheral lymphocytes in each group was obtained by using the lymphocyte separation solution,RNA extraction kit and the first chain c DNA synthesis kit.(3)Through the application of Specific internal and outer coat primers for T lymphocyte gene and 2 * Taq Plus Master Mix II,we performed nested PCR amplification and agarose gel electrophoresis for the cDNA stoste and obtained the target fragment.(4)Using high-throughput sequencing(HTS)technology to obtain the distribution of V gene,J gene,VJ gene pair(CDR3 sequence)and CDR3 amino acid(AA)sequence,respectively,to establish TCR CDR3 immune repertoires.(5)The acute myocardial infarction patient group and the healthy control group were compared to investigate the diversity of TCR CDR3 immune repertoire and amino acid length distribution in patients with acute myocardial infarction,and to find common specific high frequency expression sequences.Result:(1)The WBC,NEUT%,LY%,and MONO% in the blood of patients with acute myocardial infarction were changed compared with the healthy control group.Compared with the healthy control group,the WBC and NEUT% in patients with acute myocardial infarction were significantly higher than those in healthy controls(P<0.01);LY% was lower than that in healthy controls,with statistical significance(P< 0.01);MONO% had no significant change compared with healthy control group,and no statistical significance(P>0.05).(2)The LY% in patients with acute myocardial infarction generally showed a decreasing trend with the increase of high-sensitivity troponin I,but there was no significant correlation(P=0.2273).(3)The diversity distribution of V gene,J gene pie chart and VJ gene diversity in three-dimensional histograms in patients with acute myocardial infarction were all lower than those in healthy controls,and the histogram of amino acid length in CDR3 was skewed.At the same time,the V gene,J gene,and CDR3 sequence types were also decreased compared with the healthy control group,and statistically significant(P<0.05).(4)Ten patients with acute myocardial infarction also had the same group of specific high frequency expression sequences,namely TRB9/TRBJ2-2/CASSGGVTGELF,and were significantly higher than those in healthy controls,with statistical significance(P<0.01).Conclusion:(1)Compared with the healthy control group,the diversity of the TCR CDR3 immune repertoire in patients with acute myocardial infarction was reduced,indicating that the diversity of the immune library is closely related to the occurrence and development of acute myocardial infarction,and has a significant orientation for the early diagnosis of acute myocardial infarction.(2)The same group of specific high-frequency expression sequences existed in the TCR CDR3 immune repertoire of peripheral blood of patients with acute myocardial infarction,namely TRB9/TRBJ2-2/CASSGGVTGELF,suggesting the presence of specific antigens in acute myocardial infarction diseases.It provides theoretical basis and molecular target for immune intervention therapy.