| BackgroundsThe pathogenesis of aplastic anemia (AA) is complicated. Previous research showed that (1) the intrinsic proliferation and differentiation deficiency of hematopoietic stem cell (HSC),abnormity of hematological microculation, and disorder of immunity were involved in AA disease, insulting in hemopoiesis failure. However, recent reports indicated that AA is an autoimmune disease which uses hematological system as a target. The maintenance of T cells function plays an important role in this process. As we know, bone marrow mesenchymal stem cell (MSC) is one kind of bone marrow stem cell which possesses the capacity of self-renewing and immune regulation. Many clinical reports showed that MSC could cure some autoimmune diseases including Systemic Lupus Erythematosus.Current research found that the specific immunophenotype of human could be the potential mechanism involving in human MSC regulating immune response . There are non-expression of MHCII and FasL , negligible expression of MHCI , and non-expression of costimulator molecules like B7-1, B7-2, CD40 and CD40L in human MSC. Therefore, human MSC has minimal immunogenicity which is hard to be recognized by immune system. In vitro experiment indicated that MSC could inhibit the proliferation of T cells in mix-lymphocyte culture or mitosis stimulation. Whereas, the immune response of T cells could not be triggered by soluble extrinsic antigens directly, but through the special pathway by the antigen presenting cells (APC). In addition, the activation and production of T cells correlates to the different type of professional APC. Among them, dendritic cells (DCs) are one of the most potent APC,which could directly stimulate the proliferation of native T cells in vivo. To date, there are few domestic reports about the influence of MSC on T cells proliferation in AA patient. Besides, no study is reported in domestic on the topic that if MSC affects the T cells proliferation in AA by DCs regulation. Therefore,study on this subject helps understanding the relevant mechanisms .Objective(1)To explore whether MSC has inhibiting effect on the proliferation ofAA patients' Tcell;(2)To discuss whether MSC affects T cell's proliferation via adjusting the growth of DCs.Contents(1) After co-culture of MSC from normal bone marrow and T cells from AA patients, the differences of T cell surface antigen and subpopulation were determined by flow cytometry before and after cell co-culture,respectively. Besides,the expression of immune relevant cytokines such as IL-2,IL-4,IL-10,IFN-γwere also analysis by ELISA to study the effect of MSC on the T cell activation and proliferation in AA patients.(2) DCs was induced by normal human peripheral blood mononuclear cells,followed by the co-culture of immature DCs under LPS stimulation and with or without MSC from normal human bone marrow. To study the influence of MSC on DCs development, the expression of DCs surface antigen,costimulatory molecule CD80 and antigen CD83 in the co-culture treatments with or without MSC were determined by flow cytometry,respectively.(3) DCs was induced by normal human peripheral blood mononuclear cells,and then was stimulated by LPS to get the mature DCs. After co-culture of mature DCs and with or without MSC from normal human bone marrow,the expression of DCs surface antigen,costimulatory molecule CD80 and antigen CD83 were analyzed by flow cytometry to study the influence of MSC on mature DCs.Materials and Methods(1) MSC were separated and cultured in vitro. Cell morphology was observed and the cell surface antigen was determined by flow cytometry.(2) Peripheral blood mononuclear cells were extracted from 20 patients suffered AA and then T lymphocytes were separated by nylon fiber column. Flow cytometry was applied to determine the surface antigen and subpopulation of T cell. (3)mononuclear cells were separated from normal human peripheral blood. DCs were prepared under the culture condition of recombination human granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human interleukin-4 (IL-4). After acquiring the mature DCs induced by LPS,the phenotype analysis of DCs before and after culture was examined by flow cytometry,respectively.Results(1) After co-culture of MSC and T lymphocytes from AA peripheral blood, flow cytometry showed that the ratio of D8+ in T cells reduced significantly from 38.7% to 29.7 % (p < 0.05), whereas the CD4+ ratio increased from 24.9% to 34.9% significantly (p < 0.05). Meanwhile,ELISA analysis indicated that the concentration of IL-2 and IFN-γwere significantly decreased from 38.9 and 38.5 ng/L to 6.8 and 6.6 ng/L,respectively (p < 0.05). However,IL-4 and IL-10 increased from 2.8 and 2.9 to 5.3 and 8.3 ng/L, respectively (p < 0.05).(2) After the induction of immature DCs by LPS,flow cytometry showed that the expression of CD1a increased from 2.4% to 68.4% in the treatment without MSC,while that of CD14+ decreased from 83.6% to 3.5% (p < 0.05).(3) After the co-culture of mature DC and MSC, the expression of CD14+ increased from 5.8% to 62.8% when the expression of CD1a,CD83 and CD80 decreased from 48.6%, 60.8% and 50.2% to 30.7%,40.9% and 20.3%,respectively.ConclusionsOur study shows that (1)MSC inhibits the proliferation of T lymphocytes from AA p-atients by regulating CD8+ and CD4+; (2)futher study indicates that MSC can inhibit the growth of DCs and reverse the status of DCs from matureness to immatureness; (3)thus su-ggests the possible mechanism of MSC's inhibiting effect as follows: MSC decreases the liveness by controlling the growth of DCs and further inhibits its proliferation.
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