Diversity Of TCR ?-chain Of CDR3 Repertoire In Liver Transplantation Recipients During Perioperation

Hepatitis B in decompensated liver cirrhosis is a common chronic liver disease,which mainly due to hepatitis B virus(HBV)that persistently infect in patients and repeatedly acts on the lesion,leading to liver cell severe damage,apoptosis and so on.It can cause a series of physiological and pathological changes that appear some clinical characteristics,such as jaundice,esophageal varices,ascites,hepatic encephalopathy(HE)and spleen enlargement and so on.However,its etiology and pathogenesis is not clear,it is recognized be related to ge netic,environmental triggers and immune system disorders.At present,the ultimate cure for end-stage liver cirrhosis is liver transplantation,but the number of transplants accumulate,the rejection reaction,resistance and other complications after liver transplantation still can't avoid,of which the immune problems become the main factors that influence the postoperative survival rate.In recent years,with the development of the molecular biology and medicine and sequencing technology,more and more researchers paid more attention on immunological study,of which research approach is becoming more perfect and mature,from flow cytometry,immune scanning spectrum analysis technique and so on to the development of a new generation of sequencing technology in view of the immune cells.It means that the immunology research has entered a new period.But how to use sequencing technology to solve the immune related problem,more researchers will focus on it.The immune repertoire diversity can be analyzed through the establishment of the immune repertoire of hepatitis B in decompensated liver cirrhosis patients for Pre-and Post-liver transplantation and help us better understanding of the pathogenesis of the disease and improving the level of prevention and diagnosis of related diseases.Objective:We applied the next generation sequencing(NGS)to analyze the peripheral blood T lymphocyte immune repertoire of hepatitis B in decompensated liver cirrhosis patients for perioperative period.The diversity of T lymphocyte immune repertoire in end-stage liver cirrhosis with hepatitis B for Pre-and Post-liver transplantation will be researched for understanding the immune state of the patients,monitoring the disease process of Post-liver transplantation and guiding pharmacy.Meanwhile,it will provide a new basis for early diagnosis of hepatitis B liver cirrhosis and prognosis and therapeutic effect.Methods:We collected 6 cases of patients peripheral blood sanples that collected from liver transplantation before 1days,after 1 and7 days in 181 st hospital organ transplantation and dialysis center of PLA and 6 cases normal control groups from medical center.Peripheral blood(5 mL)was collected from venipuncture into an ethylenediaminetetraacetic acid(EDTA),and than the whole genome DNA was extracted.The primers for the region of V family and J family was used to amplified the whole CDR3 area of T cell receptor(TCR).Combined with high througlput sequencing platform,the egression frequency of each gene family,V-J pairing,base insertion/deletion,the region of CDR3 diversity and length distribution characteristics of T cell TCR beta chain in peripheral blood T lymphocytes of Pre,Post1,Post7 and NC group were analyzed.Finally,the highly clonal amplification DNA sequence,amino acid sequence(AA)and V-J combination in Pre,Post1,Post7 and NC group were screen.In addition,we analyzed the TCR diversity and differentially expressed clones according to the Distinct(uniq)clone number,Simpson and Shannon Weiner coefficient to reveal the immunological characteristics of T lymphocytes in Pre,Postl,Post7 of Hepatitis B in decompensated liver cirrhosis and NC group,to elucidate the pathophysiological mechanism of Hepatitis B in decompensated liver cirrhosis and find new ideas and new methods.Results:1.The frequency of length distribution:the distributions ofCDR3,VD indel,and DJ indel lengths were similar among the Pre,Post1,Post7 of Hepatitis B in decompensated liver cirrhosis and NC group.The 5 most common observed DJ indel lengths were 0,2,3,1 and 4nt,the 5 most frequently observed CDR3 lengths were 42,45,39,36 and 48nt,the 5 most frequently observed VD indel lengths were 0,2,3,1 and 4nt.2.Clonal amplification degree:Compared with the normal control group,the clonal amplification degree of the Pre,Post1,Post7 group was general increased.3.Highly expanded clones(HEC):After statistical analysis,we screened the HECs in Pre,Post1,Post7 group of Hepatitis B in decompensated liver cirrhosis and NC group,at the resolutions of DNA sequence(43,41,14 and 50),AA sequence(43,41,14 and 49)and V-J combination(292,299,304 and 232).In addition,the frequency distribution showed that the majority of the repertoire comprised a small number of HECs and also showed a right-skewed distribution in which the majority of the clones were low frequency.4.TCR diversity:Through calculating the diversity coefficient,Sirrpson coe fficient of Pre,Postl,Post7 group of hepatitis B in decompensated liver cirrhosis and NC group,statisticing the significant differences.We found that the immune diversity in Pre,Postl,Post7 of patients with hepatitis B end-stage liver cirrhosis was significantly lower than that in normal control group.Conclusion:1.Compared with the normal control group,the degree of T cell clonal expansions in the Pre,Postl,Post7 group was higher,and the immune diversity of Pre,Postl,Post7 group patients were lower than the control group.2.Immune repertoire sequencing can reflect the diversity of immune repertoire that through the dynamic change characteristics comprehensive analysis of the diversity in the T cell receptor beta chains complementarity-determining region 3(CDR3)of hepatitis B in decompensated liver cirrhosis patients which calculated at different resolutions of distinct DNA sequence,amino acid sequence,and V-J combination,can deeply understand the immune state of the patients,monitoring the disease process of Post-liver transplantation and guiding pharmacy.