Study On The TCR Rearrangement And CDR3 BV Spectra And Molecular Characteristics Of Ankylosing Spondylitis

Objective:To analyze CD4+and CD8+T cells lineage polymorphism of 26 TCR BV CDR3 in peripheral blood of patients with ankylosing spondylitis(AS) and detect genes that relate to TCR gene rearrangment,Exploring TCR CDR3 length and TCR Gene Rearrangement relate with AS.Provide a new pathway to study pathogenesisof T cells and personalized treatment of AS.Methods:(1) Peripheral blood mononuclear cells(PBMC) from the patients with AS and the healthy donor were sorted into CD4+and CD8+T subsets,using monoclonal antibody-coated magnetic beads.(2) Total RNA was extracted from CD4+ and CD8+ T cell subsets using RNA isolation system,and then the total RNA was used as temolate for c DNA synthesis according to RT-PCR Kit instructions.(3)Design and synthesis of primers of TCR of β chain variable gene and experimental control primers,PCR amplification of all samples TCR BV CDR3 subfamilies.Identified products by 2% agarose gel electrophoresis.(4) The profiles of TCR BV CDR3 in CD4+,CD8+ T subsets were assayed using immune scanning technique(Gene Scan)and sequencing the monoclonal/oligoclonal subfamilies,analysis clonal lineages and characteristics in CD4+and VD8+TCR BV subfamilies.(5) detect the expression of RAG and Td T,Ku70,Ku80 m RNA by RT-PCR method,and detect the sj TRECs by nested PCR method.Results:(1)In a normal controls and patients of TCR BV lineages expression on 2% agarose gel electrophoresis.In a normal controls of CD4+TCR BV and CD8+TCR BV subfamilies showed videlicetly distribution by Gene Scan.(2) Most CD4+T cells TCR BV CDR3 scanning spectrum of ankylosing spondylitis patients showed Gauss distribution is similar to the normal controls, a few CD4+ T cells TCR BV CDR3 showed skewing peak or a irregular abnormal peak and a rare CD4+ T cells TCR BV CDR3 showed oligoclonal trend and monoclonal,only two patients show a oligoclonal peak. The rate of different subsets of abnormal peaks is between 4%(1/26)and 27%(7/26) with different patients,abnormal peak high frequency families were TCR BV 2and BV3(75%,9/12),BV 19(50%,6/12).TCR BV 9 and BV 13.2abnormal peak frequency of the lowest,only 8.3%(1/12).(3) Most spectral type of CD8+TCR BV CDR3 showed abnormal distribution peak with 12 AS patients, especially to the CD8+TCR BV subfamilies of No.7 showed over 50% abnormal distribution peak,it is more than CD4+ Tell.All of 26 TCR BV subfamilies showed abnormal peak rate, abnormal peak high frequency families were BV 3 and BV19(83%,10/12),TCR BV 8 subfamily abnormal peak frequency of the lowest showed 17%(2/12).(4)In the same patient, the BV CDR3 BV TCR region length and sequence were different. The length and sequence of the CDR3 region of the same BV subfamily were different in different patients. Show that there are some common base sequence.(5) CD8+ TCR BV subfamilies of 6 AS patients showed abnormal peak rate more than CD4+ TCR both before and after treatment. There are 3 CD4+ and 4 CD8+TCR of patients showed lower abnormal peak rate after treatment.(6)20 cases of healthy controls, cases in the control group and atherosclerosis patients RAG1, RAG2, TDT, Ku70, Ku80 PCR product in 2% agarose gel electrophoresis shows, ankylosing spondylitis(as) patients and normal healthy control group showed the expression of Ku70; 8 cases of patients with ankylosing spondylitis(as), 3 cases of normal healthy detected Ku80 specific bands. Other bands were not detected.(7) sj TRECs S1 BD1-BJl were detected in 16 cases of normal controls,and no sj TRECs were not detected in patients with AS.Conclusions:(1)The CD4+ and CD8+TCR BV CDR3 lineage have significant characteristic polymorphism in AS, CD8+T cells pay a major role in the immune pathogenesis of AS.(2) Monitore the CD4+ and CD8+ TCR BV CDR3 spectrum of AS patients before and after treatment, it is possible to become a new effective technical means to understand the patient’s condition and indirectly reflect thecurative effect and prognosis of the patients.(3) TCR rearrangements were not dectected in peripheral TCR of AS patients, and thus the TCR rearrangements may not be one of the reasons that lead to the abnormal changes of TCR BV CDR3 that found in abnormal changes of BV TCR in AS patients.