| Objective:Chronic immune thrombocytopenia(ITP)is an autoimmune disease characterized by an increased risk of platelet destruction and bleeding.The pathogenesis is not clear,humoral immunity and cellular immunity is the main pathogenesis.Dendritic cells(DCs)are antigen-presenting cells that induce a primary immune response.DCs plays an important role not only in inducing a primary immune response but also for inducing immune tolerance and regulating T cell-mediated immune responses,and DCs exhibit abnormal immunological characteristics in other autoimmune diseases.To investigate the difference of the biological characteristics and function of costimulatory molecules CD70 between DCs and normal human DCs in chronic ITP patients,and to provide theoretical basis for the pathogenesis of CD70 in chronic ITP patients and to explore the pathogenesis of chronic ITP patients.Methods:(1)Dendritic cells(DCs)were generated from monocytes isolated from peripheral blood,the immunophenotype markers were detected by flow cytometry.(2)Chemically synthesized siRNA were transfected into the cells by Lipofectamine 2000 to choose the most effective siRNA to block CD70 expression in three candidates.(3)The mRNA expression and protein synthesis were analysed by real-time RT-PCR and flow cytometry.(4)CD4+ T cells were co-cultured with DCs to assess the suppression capacity of DCs in the proliferation of CD4+T cells and the production of IL-10 and IFN-γ by CD4+T cells.(5)CD4+T cells were co-cultured with DCs to assess the ability of DCs in the induction of tregs.Results:(1)DCs from ITP patients,dendrites and dense more obvious compared with normal.There is no significant difference in surface markers.(2)Dendritic cells were transfected with FITC-labelled scrambled siRNA for 4-6h.The transfection efficiency was average 60%by evaluation.(3)After transfection(48 h),real-time RT-PCR analysis showed strong and specific downregulation of CD70 mRNA levels in transfected 293T cells(p=0.046,siRNA1\2\3 Inhibition rates(63.6 ± 8.5)%,(69.0 ±11.7)%,(86.1 ± 3.2)%,but not in negative control or in the absence of siRNA.(4)CD70-3 siRNA was extraordinarily effective in repressing the expression of CD70.The expression of CD70 on the DCs with siRNA3 was also significantly lower,but showed no difference in negative control.(5)DCs from transfected group could inhibit the proliferation of PHA-activated CD4+ T cells than the negative control in ITP patients,but showed no difference in normal control group.(6)In chronic ITP patients and normal controls,DCs from transfected group could inhibit the expression of IFN-y compared to the negative controland increase the expression IL-10.(7)In chronic ITP patients and normal controls,DCs from transfected group were defective in inducing the CD4+T cell differentiating to CD4+CD25+CD1271ow Tregs compared to the negative control.Conclusion:In our study,DCs from ITP patients were significantly dense and denser than normal control,suggesting a potential role for DC hyperactivity in patients with ITP.siRNA is capable of inducing RNAi in DCs,it can knock down CD70 gene expression specifically and effectively.DCs from transfected group could inhibit the CD4+T cells and induce the CD4+T cells differentiating to Tregs.This approach is a useful tool by which costimulatory molecules of DC can be studied as well as a potential therapeutic option for autoimmune disease.Objective:ITP(Immune thrombocytopenia)is an autoimmune disease characterized by platelet decrease and risk of bleeding increase.Usually CD83 is mature dendritic cells selective marker,but also found in other kinds of cells including thymic epithelial cells an T-cells,especially regulatory T cells and B cells.CD83 and sCD83 may play an important role in autoimmune.In our research,we mainly studied three questions.Firstly the CD83 expression in ITP patients’ CD4+T cells.Secondly the effects of classical CD3/CD28 signal and TGF-β stimulation to CD83 expression in CD4+ T cells.Finally CD4+ T-cells differentiate CD4+CD25+FoxP3+ inductive T-cells.The aim of the research is to explore the CD83 expression in CD4+ T-cells subset and analysis the relationship between CD83+ and ITP patients’ status and prognosis.Methods:1.We enrolled 76 ITP patients,of which including 21 untreated ITP patients、28 active ITP patients and 27 patients in remission,and 20 healthy controls.2.Tested ITP group and control group the time-depened relationship respectively through FCM;tested the CD83+CD4+T cells expression in different ITP group and normal group and done correlation analysis with clinical indexes,such as gender,age,progress and platelet count.3.PBMCs of ITP and normal group were cultured in vitro.anti-CD3/28,anti-CD3/28+TGF-β,anti-CD3/28+TGF-β+CD83 monoclonal antibody taken into culture system respectively,RT-PCR was used to test CD83 expression in PBMCs and FCM was used to analyze the relationship between CD83 and CD4+CD25-、CD4+CD25+、CD4+CD25+FOXp3+Treg.4.ELISA was used to test the concentration of sCD83 in plasma,then made the correlation analysis between sCD83 and platelet count.Results:1.the FCM showed the ratio of CD83+CD4+T cells as follows:initial treatment group(6.47%±0.55%),active stage group(3,93%%±0.46%),remission group(2.97%±0.26%),normal control group(2.60%±0.33).Initial treatment group and active stage group were higher than remission group and normal control group significantly.There is statistical significance(P<0.01).2.By the stimulation of CD3/CD28,all groups showed time depending.ITP groups expressed few CD83+CD4+T cells at first,then CD83+CD4+T cells positive percent began to increase in first day(2.49±0.31%),remarkable increasing in second day(5.48±0.48%),however decreased in third day(1.97±0.32%).3.In all groups,CD4+CD25-cells expressed low CD83,CD4+CD25+ cells higher,CD4+CD25+FOXp3+Treg cells highest.4.AntiCD83 decreased the expression of CD83.TGF-β increased the expression of CD83.Compared with normal group,ITP groups express more CD83 mRNA.In accord with CD83+CD4+T cells,ITP groups’ sCD83 in peripheral plasma increased,however negative correlation with platelet count(r=-0.438,p=0.007),no correlation with gender and age.Conclusions:1.CD83+CD4+T cells was significantly negative correlation with ITP stage.2.Platelet count was significantly negative correlation with sCD83.3.As the release of illness,CD83+CD4+T cells expression and sCD83 level down significantly.4.CD83 expression increasing in Treg cells may connected with immune suppression function disorder,at the same time sCD83 may monitor the status of ITP patients in the future. |
PDF Full Text Request