| Objective:To explore mechanism of human bone mesenchymal stem cell(MSC)in treating patients with aplastic anemia(AA).Methods:(1)MSCs in patients with aplastic anemia(AA)and the control group were separated with Percoll(1.073 g/m L)and cultured in low glucose DMEM.T cells were harvested by using nylon column.Then,observing their morphologies,checking their molecule surface antigen by flow cytometry and examining the process of adipogenic differention by RT-PCR.(2)The mononuclear cells(MNC)of marrow in patients with AA were enriched based 1.077g/l density centrifuge and cultured on the 1640 medium containing 100 ml/L fetal bovine serum.Further,we established groups by pathogenesis in 56 patients with AA.(3)①MSC in control group and MNC in AA group were co-cultured with or without cytokines.The function of supporting hematopoiesis for MSC was to be observed in single confluence layer after plating by counting the total cells and the clones in every well every week.Then analyzing the dynamics of proliferation②MSC in control group and MNC in AA group were co-cultured with or without cytokines.The proliferation of T cell was measured by MTT method.The CD25(IL -2R),CD69,CD4,CD8,Annexin-Ⅴexpression rates of CD3+Tcells were analyzed by flow cytometry.the gene and protein of IL-2,IL-4,IL-10,TNF-α,IFN-γ,TGF-β1 were examined by RT-PCR and ELISA respectively.(4)MSC treated to the model of AA,by the examination of peripheral hemogram,bone marrow biopsy,pathological section of spleen. Results:1,(1)there was no significant difference between control group MSC and AA-MSC in morphologies.(2)AA's bone marrow stromal cells(AA-BMSCs)are the potential IL-15 expressing cells and the basic constituting elements of bone hematopoietic microenvironment.(3)adipogenic differentiation in AA patients is earlier than controls.2,the defective bone marrow AA,the defective stem cells AA and immune-mediated AA is 24.1%,28.0%and 44.8%respectively.3,The clones of CFU-GM in group 1(MSC)(78.46±3.58)/2×105cells,after 14 days cultured was and significantly higher than(9.21±4.32)/2×105cells in group 3(CK DMEM medium),while lower than(99.32±4.34)/2×105cells in group 3(MSC+CK).4,(1)the Treg cells(TCD4+CD25+)in AA group(2.01±1.21)/2×105was significantly lower than (4.43±1.67)/2×105cells in control group,while(5.43±2.31)/ 2×105in group 3 (MSC+AAT)was no more than(4.43±1.67)/2×105cells in control group.(2)It was that CD25 and CD69 in T cells of AA was long-term expressed exposed aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction.(3)MSCs significantly inhibited T cell proliferation(P＜0.01). (4)flow cytometry analysis discovered that MSC increase CD4 and decrease CD8, But the expression of Annaxin-Ⅴhad no significant difference compared with control group(p＞0.05).(5)RT-PCR and ELISA analysis showed that MSCs induced the expression of IL-4,IL-10,TGF-β1 and decreased significantly the expression of IL-2,TNF-α,IFN-γin T cells of AA.5,(1)the model of AA formed by injection 50.0 mg /kg chloramphenicol and 62.5 mg/kg cyclophosphamide for three days,on the fourth day after the irradiation with 3.0 Gy 60Co- Y rays.This model had the following characteristics:easy to be operated and be carried out,reproducible,steady,pathological mechanism similar to clinical AA,lasting for over 30 days and easy to screen effective drugs to treat AA.(2)By the examination of peripheral hemogram,biopsy pathological of bone marrow and spleen the mouse of AA showed improvements in 3 blood components greatly(p＜0.05).Bone marrow proliferated and restored to the normal level,hematopoietic cell increased obviously(hematopoietic cell capacity was more than 40%),and made atrophied spleen restore to normality. Conclusion:1,morphologies of AA' MSC had no evident different with the control but was more easy adipogenic differention and abnormal IL-15 expression.2,aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction.3,Human bone mesenchymal stem cells can promote hematopoiesis of aplastic anemia。4,Human bone marrow MSCs inhibited T cell activation and proliferation in patients with AA in vitro.5 The fundamental mechanism of MSC in treating AA should be potential to promote hematopoietic cell proliferation and adjust immunity of model mouse. |
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