Combinatory Effect Of Rapamycin And Cyclosporin A On Hematopoiesis In Mice With Immune-Mediated Aplastic Anemia

Objective：To observe the hematopoiesis in the context of combinatory administration ofRAPAmycin and cyclosporin A in mice with immune-mediated aplastic anemia andexplore a novel paradigm of AA treatment.Methods:Female Kunming mice (receptor) were radiated with sublethal dose（6Gy）ofX-ray and then accepted the1×106lymphocytes from Balb/c mice (donor) byintravenous injection within4hours to establish the AA models. All mice weredivided at random into five groups.1. RAPA~+CsA:A:RAPA(0.1mg·kg-1·d-1)~+CsA(5mg·kg-1·d-1),for5days;B:RAPA(0.5mg·kg-1·d-1)~+CsA(5mg·kg-1·d-1),for5days;C:RAPA(1mg·kg-1·d-1)~+CsA(5mg·kg-1·d-1),for5days;2.RAPA group:RAPA(1mg·kg-1·d-1)) for5days;3. model group:immune-mediated aplastic anemia mice without any treatment;4.CsA group:CsA(5mg·kg-1·d-1)),for5days;5. normal control group: normal mice;The general status, peripheral blood cells, GM-CFU and nucleated cells of bonemarrow in different times were observed in mcie from various groups. Before thedeath of mice, thigh-bones were taken to exact bone marrow. The bone marrows wereobserved pathologically with slice techniques and hematoxylin and eosin stain.Theexpression of CD3~+,CD3~+CD4~+,CD3~+CD8~+,CD4~+CD25~+in T-cell from spleenwere examined by FCM.Results:1. The leukocytes and platelets in mice from AA group began to decrease afterthe model establishment with the minimum level at the eighth to fourteenth day (WBC:0.42×109/L, PLT:3.50×109/L). No mice were alive at the14th day. Within thesuppressed bone marrow, the reduction of hematopoietic cell accompanied theincreasement of adipocytes in the medullary space. FCM detection showed thatCD4~+CD25~+T cells decrease（CD4~+CD25~+:24.30%and the CD3~+, CD3~+CD8~+,CD3~+CD4~+T cells increased significantly in spleen (CD3~+:86.85%, CD3~+CD8~+:53.48%, CD3~+CD4~+:63.14%).2. The hemopoiesis in RAPA~+CsA group (B and C) improved markedly.At the14thday since model establishment, the survival rate of mice in Group Band C was70%and60%respectively, higher than that in AA group.In comparison with AA group, the numbers of WBC and platelet in mice of thetreat group began to rise from the2ndweeks. Also the numbers of nuclear cells andCFU–GM in double thigh-bone marrow increased significantly（Group B：8.42×106，50.20.Group C：7.79×106，48.20）. Bone marrow pathological section showed theactive hyperplasia of bone marrow and increasing hematopoietic cell with a fewadipocytes in the medullary space. The expressions of CD3, CD3~+CD4~+andCD3~+CD8~+T cell in Group B and C were lower than that in AA group. No significantdifferences were observed between the control group and Group B and C（Group B：p=1.00, Group C：p=0.672,＞0.05）.3. Higher expressions of CD4~+CD25~+T cells of spleen in RAPA~+CsA group (C)and RAPA group were found than that in model group. No significant differenceexisted between the control group and RAPA~+CsA group (C) and RAPA group（Group C: p=0.29, RAPA group：p=0.995,＞0.05）.RAPA treatment was found toincrease the number of CD4~+CD25~+T cells in a dose-dependent manner.Conclusions:1.At the fourth hour after radiation with sublethal dose（6Gy）of X-ray, Kunmingmice accepted the infusion of1×106lymphocytes from balb/c mice by intravenousinjection, which successfully establish the model of immune-mediated aplasticanemia.2. Rapamycin has strong immunosuppressive activity, the comination of RAPAand CsA have synergistic action for restraining T cells’ activity in mice with immune-mediated aplastic anemia. The combinatory administration of RAPA andCsA can improve hematopoiesis in mice with immune-mediated aplastic anemia viarebalancing the immune disturbance, suggesting a novel paradigm in treating patientswith AA.3. Rapamycin can promote the proliferation of CD4~+CD25~+T cells in mice withimmune-mediated aplastic anemia.