Effect Of Low-dose Cyclophosphamide And Cyclosporin A On Hematopoiesis In Mice With Immune-mediated Aplastic Anemia

Objective:To observe the effect of low-dose cyclophosphamide and cyclosporin A on hematopoiesis in mice with immune-mediated aplastic anemia and explore a new method of treating aplastic anemia.Methods:Female Balb/c mice were used as donor, female kunming mice as receptor. The receptors were radiated with sublethal dose (6Gy) of X-ray,then 1 X 106 lymphocytes of the donors were infused by intravenous injection within 4 hours to establish the models.A total of 150 mice were divided randomly into the test groups and the control group:1.administration of different times:to inject drugs on the dO, d4 and d7 after establishment of model;2.administration of different drug concentration:1)LD-CTX+CsA:A:CTX(10mg·kg"1-d-1)+CsA(5mg·kg-1-d-1),for 5 days;B;CTX(20mg·kg-1-d-l)+CsA(5mg·kg-1-d-1),for 5 days;C:CTX(30mg·kg-1-d-1)+CsA(5mg·kg-1-d11),for 5 days;2)HD-CTX group:CTX(50mg·kg"-d-1),for 4 days;3.model group:immune-mediated aplastic anemia mice without any treatment;4.CsA group:CsA(5mg/kg/d),for 5 days;5.control group:normal mice without any treatment;The change in peripheral blood cells of mice and generral conditions was observed in all groups. When mice were on the brink of death (or at 28 days after establishment of model), thigh-bones were taken and pathological examination of bone marrow was carried out. The bone marrow was taken to be incubated in mythyl cellulose semisolid culture system for observing proliferation of CFU-GM. CD3+,CD3+CD4+,CD3+CD8+,CD4+CD25+, Foxp3+Tcells and Thl/Th2 helper T cells of spleen lymphocytes were detected through FCM.Results:l.The leukocytes and platelets of mice in model group decreased after establishment of model and achieved the lowest level after 8-14 days.CD3+ CD3+CD8+T cells and IFN-y of spleen lymphocytes detected through FCM significantly increased.CD4+CD25+and Foxp3+Tcells decreased.Bone marrow hyperplasia was depression.The number of hematopoietic cell was decreased and a lot of adipocytes was in the medullary space.2.Hemopoiesis of LD-CTX+CsA group(A,B) improved.The survival rate of the mice at 28 days after establishment of model was higher than model group.Compared with model group, in the treated groups mice's WBC and platelet count were rising from the second week on. And the numbers of nuclear cells in a single thigh-bone and CFU-GM were increased significantly. Bone marrow pathological section showed that bone marrow hyperplasia was active, the number of hematopoietic cell was increased and the number of adipocytes was small in the medullary space. Compared to model group:these groups lower expressed CD3+,CD3+CD4+and CD3+CD8+Tcells of spleen lymphocytes;group A highly expressed CD4+CD25+, Foxp3+T cells of spleen lymphocytes.The ratio of Thl/Th2 helper T cells of group A was significantly lower than the model group.but in group B the ratio was no statistically significant.3.Hemopoiesis of HD-CTX group didn't improved.The dath rate of this group was high.Bone marrow pathological section showed that Bone marrow hyperplasia was depression.The number of hematopoietic cell was decreased and a lot of adipocytes was in the medullary space.Conclusions:1.Kunming mice were radiated with sublethal dose (6Gy) of X-ray, then 1 X 106 lymphocytes of balb/c mice were infused by intravenous injection within 4 hours.This can establish the models of immune-mediated aplastic anemia.2. The immune function of cyclophosphamide is related with dose.HD-CTX can inhibit immune function;LD-CTX can inhibit and regulate immune function.LD-CTX dosen't injury hemopoietic stem cell.3.LD-CTX and CsA can correct immune disturbance and promote hematopoiesis.