| Objectives:By analyzing the effect of human bone marrow-derived mesenchymal stem cells(MSCs)on ratio of CD4~+CD25~+ T cells and secreting interleukin-2 (IL-2),interferon-γ(IFN-γ),IL-4 and IL-10 from T lymphocytes of ITP,and furthermore the relationship between the two types of effect in vitro,we could study the mechanism of correcting immune balance and inducing autoimmune tolerance of ITP by MSCs,so as to offer preliminary experimental data for clinical treatment of ITP.Methods:Human bone marrow- derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells,and the purity of MSCs was identified with the spindle-fibroblastic morphology characterized by microphotograph and the phenotypes tested by flow cytometry,respectively.Allogeneic T lymphocytes of health adults and ITP patients were isolated from peripheral blood by Ficoll Hypaque and nylon cotton column,and their ratio of the CD4~+CD25~+ T cells was detected by flow cytometry.Then the stromal feeder layers of different amount(2×10~3,1×10~4, 5×10~4)per well of MSCs dealed with mitomycin were cocultured with above-mentioned T lymphocytes,and T lymphocytes and supernatant were collected on day 2,4 and 6 after cocultivation,respectively,then the level of IL-2,IFN-γ,IL-4, IL-10 secreted by T lymphocytes was measured by enzyme linked immunosorbent assay(ELISA),and the ratio of CD4~+CD25~+ T cells in every experimental group is detected by flow cytometry.Results:Mononuclear cells isolated from bone marrow adhered the bottom of the flask and were fusiform shape.The primary cell grew as many colonies,and reached confluence in about 10 days.The passage cells had the same morphology feature as the primary and grew quickly that reached confluence in 6 or 7 days.According to the flow cytometry assay,MSCs were uniformly positive for CD29,CD105 and CD166, while negative for CD34,CD45 and HLA-DR,and the purities of MSCs were above 95%from the third passage on;furthermore,the purities of the T lymphocytes isolated by nylon cotton column could reach 70%～80%.The result determined by flow cytometry assay as follows:the amount of CD4~+CD25~+ T cells and the ratio of CD4~+CD25~+/CD4~+ in the peripheral blood of ITP patients was obviously lower than normal control(P＜0.05,respectively).While the amount of MSCs exceeded 1×10~4 and cocultured with T lymphocytes with PHA-activating for 4 days,MSCs could obviously up-regulate the ratio of CD4~+CD25~+ T cells and significantly increase CD4~+CD25~+/CD4~+ in the peripheral blood of ITP patients and health adults compared to control(P＜0.05,respectively)in a dose dependent way(P＜0.05,respectively).IL-2,IFN-γ,IL-4 and IL-10 had not been secreted by MSCs themselves detected by ELISA.The level of IL-2 and IFN-γ,secreted by PHA-activating T cells from ITP patients was higher than normal control(P＜0.05,respectively),inversely,IL-4 and IL-10 was lower than normal control(P＜0.05,respectively).After cocultured with PHA-activating T lymphocytes,MSCs could significantly inhibited the cytokine level of IL-2 and IFN-γsecreted by T lymphocytes of ITP patients or health adults(P＜0.05, respectively)in a dose dependent way(P＜0.05,respectively),and the effect was higher when co-cultured for 4 days and 6 days than that for 2 days(P＜0.05, respectively);however,MSCs could significantly promoted IL-4 and IL-10 secreted by T lymphocytes of ITP patients(P＜0.05,respectively)in a dose dependent way (P＜0.05,respectively),while the effect on IL-4 had no obvious correlation with cocultivation-time(P＞0.05),but the effect on IL-10 in a time dependent way(P＜0.05). While their amount exceeded above 1×10~4,MSCs could obviously promoted IL-4 level secreted by T lymphocytes of health adults(P＜0.05)in a dose dependent way(P＜0.05),also they could obviously promoted IL-10 level secreted by T lymphocytes of health adults(P＜0.05)in a dose dependent way(P＜0.05).And both of the effects were higher when co-cultured for 4 days and 6 days than that for 2 days (P＜0.05,respectively). As for ITP patients,whose T lymphocytes and CD4~+CD25~+T cells regulated by MSCs,though the levels of IL-2,IFN-γ,IL-4 and IL-10,and even the the amount of CD4~+CD25~+ T cells gradually tended to normal level but not reached that yet in vitro (P＜0.05,respectively).Conclusions:①ITP is a kind of Th1-dominant autoimmune diseases having abnormal T cytokines profile,and the lower quantity of CD4~+CD25~+ T cells also suggests that CD4~+ CD25~+ T cells could play an important role in the course of ITP.②By regulating the balance between Th1 and Th2 reaction as well as up-regulating the expression of CD4~+CD25~+T cells to induce immunotolerance of ITP in vitro, MSCs could play a part in use of potential immunotherapy for ITP.
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