| Objective: To screen the culture method of tolerogenic dendritic cells (tDCs) derived from bone marrow in vitro and explore the effect of infusion with tDCs on hematopoiesis in mice with immune-mediated aplastic anemia (AA). In order to offer a new way in the therapy of immune-mediated AA.Methods: Female Balb/c mice were used as donor, female kunming mice as receptor. Tolerogenic dendritic cells were generated from bone marrow precursors cultured with GM-CSF,IL-4,VIP,DXM and LPS. The cells were divided into five groups: blank group(not add any agent),common-DCs group(GM-CSF100ng/ml+IL-4 100ng/ml),VD1–DCsgroup(GM-CSF20ng/ml+VIP40ng/ml+DXM10ng/ml),VD2–DCs group(GM-CSF20ng/ml+VIP40ng/ml+DXM50ng/ml),VD3–DCs group(GM-CSF 20ng/ml+VIP40ng/ml+DXM100ng/ml). The cell morphological changes were examined under optical microscope. The differences on cell phenotype were analyzed by flow cytometry(FCM). The capability to stimulate the proliferation of lymphocytes was examined through MTT method. Forty kunming mice were divided into AA model group, tDCs group,radiation group and control group. In AA model group, every kunming mice were radiated with sublethal dose(7Gy)of X-ray, then 1×106 lymphocytes of balb/c mice were infused by intravenous injection within 4 hours. In tDCs group,1×106 tDCs were infused at 3 days and 5 days after establishment of model. The change of Mice'WBC 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. CD8+ T cells and CD4+CD25+ T cells of spleen lymphocytes were detected through FCM.Results: 1.The cells cultured with GM-CSF,IL-4,VIP,DXM and LPS possessed morphological characteristic of typical DCs. Compared to blank group, VD1-DCs group,VD2–DCs group and VD3–DCs group highly expressed CD11c(P<0.05).2.Compared to common-DCs group, VD1-DCs group,VD2–DCs group and VD3–DCs group lowly expressed CD80,CD86 and CD40, and induced light lymphocytes proliferation(P<0.05). The VD1-DCs group(VIP 40ng/ml+DXM 10ng/ml)is the most different with other groups (P<0.05).3. In AA model group, mice'WBC decreased constantly, the survival rate was 10% at 28 days after establishment of model. The number of nuclear cells in a single thigh-bone and CFU–GM was lower obviously than other groups(P<0.05). Bone marrow pathological section showed that bone marrow hyperplasia was low overly and cavitas medullaris filled with adipocytes. Compared to tDCs group and control group, AA model group highly expressed CD8+ T cells of spleen lymphocytes(P<0.05), and lowly expressed CD4+CD25+ T cells of spleen lymphocytes (P<0.05).4. The WBC of tDCs group decreased to deep canyon at 10 days after establishment of model, then gradually recovered to normal. The survival rate was 30% at 28 days after establishment of model. Compared to radiation group and control group, tDCs group was not difference obviously at the number of nuclear cells in a single thigh-bone and CFU–GM (P>0.05). It was different significantly between tDCs group and AA model group(P<0.05). Bone marrow pathological section showed that bone marrow hyperplasia was active and the number of adipocytes was small. It is similar to control group. Compared to AA group ,tDCs group highly expressed CD4+CD25+ T cells of spleen lymphocytes (P<0.05).Conclusions: 1.Tolerogenic dendritic cells were generated from bone marrow precursors cultured with GM-CSF, VIP/DXM and LPS. It is the best culture condition that VIP 40ng/ml combinant DXM 10ng/ml.2. Receptor mice were radiated with sublethal dose(7Gy)of X-ray, then 1×106 lymphocytes of donor mice were infused by intravenous injection within 4 hours. The model of immune-mediated AA is prepared.3. The infusion of tDCs in mice with immune-mediated AA can induce CD4+CD25+ T and promote hemopoiesis recovery of AA mice.
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