| Background:Chronic immune thrombocytopenia (ITP) is characterized by autoimmune-mediated platelet destruction and impairment of thrombopoiesis. The main pathogenesis of chronic ITP is the broken of immune tolerance, which result in the cellular immune and humoral immune response to the plateletes. Mesenchymal stem cells (MSCs) are proposed to exhibit immune modulatory functions in self-tolerance maintenance. The abnormal bone marrow derived MSCs had been reported in several autoimmune diseases.Objective:This study aims to assess the biological properties and functions of BM-MSCs from patients with chronic ITP.Methods:(1) Cells were isolated and purified from bone marrow by adherent culture and passaging in the culture medium (DMEM/DF12 and 10% fetal bovine serum) in vitro. Except the general culture, we also set up the group with addition supplements (AS), which contained the bFGF, EGF and ITS. (2) The morphology of BM-MSCs was observed by inverted microscope. (3) The immunophenotype markers of BM-MSCs were detected using flow cytometry. (4) BM-MSCs were induced to differentiate into adipocytes and osteoblasts cells, which were identified with corresponding staining. (5) The proliferation of BM-MSCs was detected, and the growth curve was drawn. (6) The apoptosis of BM-MSCs were detected using flow cytometry. (7) PBMCs were co-cultured with BM-MSCs to assess the suppression capacity of BM-MSCs in the proliferation of PBMCs and the production of IL-10, TNF-a and IFN-y by PBMCs. (8) CD4+ T cells were co-cultured with BM-MSCs to assess the ability of BM-MSCs in the induction of Tregs.Results:(1) We successively isolated and purified BM-MSCs from patients with chronic ITP. (2) We showed abnormal morphology of BM-MSCs from ITP patients, however, AS treatment ameliorate the appearance of BM-MSCs from ITP patients. (3) The BM-MSCs from ITP patients and normal controls showed similar phenotype:the CD73, CD90 and CD105 were positive, while the CDllb, CD34, CD45, CD14 and CD 19 were not expressed. (4) BM-MSCs from chronic ITP patients were shown to have similar capacities to differentiate along adipogenic and osteogenic lineages as those from normal controls. (5) BM-MSCs from ITP patients showed increased apoptosis compared to normal controls, while the AS treatment could down-regulated the apoptosis proportion. (7) BM-MSCs from both chronic ITP patients and normal controls could inhibit the proliferation of PHA-activated PBMCs in a dose-dependent manner, and enhanced the expression of protein IL-10, reduced the expression of IFN-γand TNF-α. The immune-inhibiting potential and the ability in promoting IL-10 secretion by PBMCs of BM-MSCs from ITP patients were defective compared to those of normal BM-MSCs. (8) BM-MSCs from the chronic ITP patients were defective in inducing the CD4+ T cells differentiating to CD4+CD25+CD1271ow Tregs compared to the control group.Conclusion:BM-MSCs from patients with chronic ITP exhibited impaired proliferation, abnormal morphology and excessive apoptosis. These defects could be ameliorated by a modified culture environment. However, the immune-inhibiting potential and the Treg-inducing ability of BM-MSCs from patients were reduced compared to those of normal BM-MSCs. Further studies will be required to determine the exact role of defective BM-MSCs play in the pathogenesis of chronic ITP.Background:Primary immune thrombocytopenia (ITP) is an autoimmune disease with many immune dysfunctions, including over-prQliferation and apoptosis resistance of auto-reactive lymphocytes. Interleukin-7 (IL-7) is a homeostatic cytokine, secreted by non-marrow-derived stromal and epithelial cells, for resting T cells with increasing serum and tissue levels during T cell depletion. IL-7 could maintain the proliferation of immature human B cells and contribute to the rearrangement of immunoglobulin.Objective:This study aimed to determine the effects of IL-7 on the cytokine production and survival of peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMNCs) from ITP patients.Methods:(1) The concentrations of cytokines in the peripheral blood plasma or in the cell culture supernatants of PBMCs were measured by ELISA. (2) The expression of IL-7Ra receptor components on the surface of T cell were detected by flow cytometry. (3) The effect of IL-7 on the proliferation of PBMCs and BMNCs were detected by the incorporation of BrdU. (4) The effect of IL-7 on the apoptosis of platelets, PBMCs and BMNCs were detected were measured by flow cytometry.Results:We found that the plasma IL-7 levels in peripheral blood from ITP patients were lower than that of the normal controls, and it had positive correlation with platelet counts. However, the levels of IL-7 did not change in bone marrow serum of ITP patients compared with that of normal controls. The result of further stimulation experiments in vitro showed that in ITP patients, IL-7 up-regulated the apoptosis of autologous platelets, promoted the proliferation and secretion of interferon-y, tumor necrosis factor-a as well as IL-10 of lymphocyte both from peripheral blood and bone marrow, suppressed the apoptosis of CD8+ T cells.Conclusion:The plasma IL-7 levels in peripheral blood from ITP patients were decreased compared to normal controls. IL-7 also showed discrepancy physiological effects on the lymphocytes from ITP patients and normal controls. As the role of IL-7 in apoptosis-resistance and stimulation of pro-inflammatory cytokines, we speculated that decreased IL-7 in peripheral blood, maybe, is a consequence of the negative feedback of the pro-inflammatory function in ITP patients.
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