| Objective: With deepened thestudy of immune rejection and the clinical application of immunosuppressive agents,organ transplantation has been rapidly developed since the twenty-first century.At present,liver transplantation has become the most principal treatment means and routine surgery to save end-stage liver disease and acute liver failure.The incidence of rejection of liver transplantation is relatively low because of liver as an immune privilege organ for solid organ transplantation,however,immunosuppressive agents have become a convenal clinical application after transplantation.Therefore,it is important to explore the relationship between the use of immunosuppressive agents and the immune status in recipitent undergoing liver transplantation and its regulation mechanism to improve the survival of patients with liver transplantation.It has been found that T lymphocytes are one of the major immune cells in the transplant rejection and immune tolerance induced by the body,and play an key role of regulation and therapy in the immune balance.T cells(Treg)as the main immunosuppressive cell subsets,specifically express Foxp3 and secrete IL-10 and FGF-b,and constitute the four major subgroups of the CD4 ~+T cells together with Th17 cells,Th1 and Th2 T cells.Th17 cells restrain with Treg cells each other by secreting IL-17,IL-22,IL-6.The dynamic balance of the Treg and Th17 plays a crucial role in the body immune defense and immune self-stabilization.However,there is limited knowledge the change characteristics of each period of long-term survival of peripheral blood lymphocyte subsets,especially the expression of Treg cells and Th17 cells of the patients after liver transplantation.In this study we examined the expression levels of CD4 ~+ CD25 ~+ Foxp3 ~+ regulatory T cells and Th17 cells in peripheral blood of patients of long-term survival with FK506(immune tolerance)after liver transplantation.in order to investigate the effects of regulatory T cells and th17 cells on immunoregulation and relationship with the survival time of the patients after liver transplantation,.Methods: The subjects of this study were the long-term survival patients after OLT from 2002 to 2015 in our hospital and healthy volunteers were as control group.Inclusion criteria: 1.The primary disease was posthepatitic cirrhosis after hepatitis B.2.The Patient received FK506 single immunosuppressive therapy.3.They didn't have acute and chronic rejection,infection,hypertension,diabetes,liver and kidney dysfunction,coronary heart disease and other complication after OLT.4.The Preoperative and postoperative patient without systemic inflammation and autoimmune diseases.5.All patients were treated with classical orthotopic liver transplantation.Healthy volunteers had no history of trauma and infection,no history of autoimmune diseases,and no history of liver diseases.Exclusion criteria: 1.There have been related complications,such as rejection,infection and so on.2.Patients with basic information incomplete and patients without follow-up.3.The patient who refused to accept clinically relevant tests.According to the above criteria,42 patients were enrolled in this study.According to the survival time of patients were divided into Tac1 group(15 patients with survival time of 6 months to 3 years after LT),Tac2 group(15 patients with survival time of 3 to 10 years after LT),Tac3 group(12 patients with survival time more than 10 years after LT).The 20 healthy individuals were assigned to control group.The peripheral blood samples of all the subjects were collected after transplantation and treated with heparin anticoagulation.The levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),albumin(ALB)and glutamyl transpeptidase(GGT)in serum were measured by automatic biochemical analyzer,Chemiluminescence microparticle immunoassay was used to determine the plasma concentration of FK506.The expression of CD4~+CD25~+Foxp3~+regulatory T cells and Th17 cells was detected by fluorescent labeled monoclonal antibody and flow cytometry.All analyses were carried out with the SPSS software(version 22.0),the measurement date was described as`x±s.One-way ANOVA was used to detect multivariate differences.The enumeration data are represented by chi-square test.A value of p<0.05 was considered statistically significant.Results:1 Indicators of liver function test(ALB?AST?ALT and GGT)were within the normal range in the long-term survival patients after liver transplantation,However,the ALT indicator changes as follows,compared with CG group,Tac1 group showed significant improvement(P<0.05),with the prolongation of survival time,the indicator decreased gradually,there was no statistical difference between the Tac2 group and the Tac3 group(P>0.05),compared with the CG group.The results showed that there was no significant clinical rejection after liver transplantation.2 Changes of T Lymphocyte Subsets in Patients with Different Survival after Liver Transplantation :The percentage of CD3~+CD4~+T cell and CD4~+ / CD8~+T cell ratio: The counts of CD3~+CD4~+T cells and CD4~+/CD8~+T cells in Tac1,Tac2,Tac3 groups were significantly lower than that in control group(P<0.05);The counts of CD3~+CD8~+T cells in the Tac3 group were significantly higher than in control,Tac1 and Tac2 groups(P<0.05).However,there existed no statistical difference in the counts of CD3~+T cells,NK cells while separately among the four groups(P>0.05).The results showed that compared with normal subjects,CD3~+CD4~+T cells were in a low level in patients with long-term survival after liver transplantation.3 Expression of CD4~+CD25~+Foxp3~+Regulatory T Cells in Patients with Different Survival after Liver Transplantation : The percentage of CD4~+CD25~+Foxp3~+regulatory T cells in CD4~+T cells in Tac1 group(4.82%±0.29%)was significantly lower than that in control group(5.66%±0.32%)(P<0.05).The percentage in Tac2 group(1.46% ± 0.29%)and Tac3 group(1.62%±0.35%)was significantly lower when compared with that in the Tac1 group(4.82%±0.29%)and control group(5.66%±0.32%)(P<0.05).However,no significant difference in the percentage of Tregs in CD4~+T cells was observed between groups Tac2 and Tac3(P>0.05).We found that the frequency of Tregs gradually decreased in peripheral blood with the survival time lengthening and maintained the relatively low level.4 Expression of Th17 cells in patients with different survival after liver transplantation :The percentages of Th17 cells in CD4~+T cells in Tac1 group(1.95%±0.33%)and Tac2 group(1.99%±0.36%)were significantly higher than that in control group(1.49%±0.36%)(P<0.05).No significant difference in the percentage of Th17 in CD4~+T cells was observed when compared to Tac1,Tac2 and Tac3 group(1.72%±0.26%)(P>0.05).And there existed no significant difference between Tac3 group and control group(P>0.05).The results show that Th17 cells in patients after liver transplantation survival increased earlier,and with the time to return to normal levels.Conclusion:1 In the case of single-dose immunosuppressive agents FK506 patients after liver transplantation,the clinical immune tolerance is good,but with lower CD3~+CD4~+ T cell level and CD4~+CD25~+Foxp3~+regulatory T cells,and higher Th17 cells,these reminds that the clinical monitoring of the patient's condition is necesarry.2 In the case of single-dose FK506 after liver transplantation,the frequency of CD4~+CD25~+Foxp3~+regulatory T cells decreased gradually with the survival time lengthening and finally maintained at a lower level in peripheral blood of patients with long-term survival3 In the case of single-dose immunosuppressive agents FK506,the expression of Th17 cells was slightly higher in the peripheral blood of patients survived 6 months to 3 years and 3 years to 10 years after liver transplantation,and the level of Th17 cells in patients who survived for more than 10 years was close to normal level. |
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