Study Of The Exosome Derived From Tolerogenic Dendritic Cells In Treatment Of The Mice With Immune-mediated Aplastic Anemia

Objective: Purification of exosome(tDex) derived from tolerogenic dendritic cells(tDcs), to explore the effect of infusion with tDex on hematopoiesis and immunoregulation in mice with immune-mediated aplastic anemia (AA).Methods: Female Balb/c mice were used as donor, female kunming mice as receptor. Tolerogenic dendritic cells were generated from bone marrow precursors which cultured with containing 10% fetal bovine serum in RPMI 1640 medium supplemented with GM-CSF20ng/ml + VIP40ng/ml + DXM10ng/ml in vitro. Supernatant were collected, exosome purified by ultracentrifugation tDex and proteins determined by Bradford method. The tDCs morphology was observed by optical microscope and the tDex by electron microscopy. Immunophenotype was examined by flow cytometry in tDCs and tDex of CD80, CD86, CD40, CD11c, Fas-L expression. The capability to stimulate the proliferation of lymphocytes was examined through MTT method. A total of 120 mice were divided randomly into the test groups and the control group:1) control group: normal mice without any treatment;2) 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.3) tDCs group: on the establishing mouse model day (d0) each Balb/c mice were injected a bonus of 1×106 tDCs through the tail vein.4) tDex group 1: in the group a, group b and group c each Balb/c mice were infused 5ug tDex , 10ug tDex and 20ug tDex respectively on the d0.5) tDex group 2 : each Balb/c mice were infused 8ug tDex on the d0, d3 and d7 after establishment of model.The change in peripheral blood cells of mice 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 +CD8+,CD3+HLA-DR+, CD4+CD25 +, Foxp3+T cells and Th1/Th2 helper T cells of spleen lymphocytes were detected through FCM. T-bet and GATA-3 gene expression were assayed by real-time fluorescence quantitative PCR.Results:1. Comparing of tDex group and tDCs group, expression of CD11c, CD80, CD86, Fas-L were not significant difference (P>0.05). Compared to tDCs group, the expression of CD40 and MHC-Ⅱin tDex group were lower (P <0.05).2. Activity of lymphocyte proliferation: lymphocyte proliferation exhibited a obvious inhibition for both tDCs and tDex group. Compared to tDCs group, activity of inhibition was higher in tDex combined with tDCs (P<0.05). In the only tDex group, the higher tDex's concentration was, the lower the activity of lymphocyte proliferation was. The group of treated by the tDex after 72 hours had a stronger inhibition than that group of treated after 48 hours. So, these suggested that the inhibition of tDex has direct proportion with time and dose.3. The mice's promote hemopoiesis of these groups that treated by infusion tDCs , 5ug tDex, and the third day infusion 8ug tDex , was obviously perfected than the model group. 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 (P<0.05). 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 highly expressed CD4+CD25+, Foxp3+T cells of spleen lymphocytes , lower expressed CD3+CD8+ T cells of spleen lymphocytes. The ratio of Th1/Th2 helper T cells was significantly lower than the model group. In these three groups, the expression of CD3+HLA-DR+ T cells of spleen lymphocytes was no statistically significant. And the ratio of T-bet/GATA-3 gene expression was significantly lower than the model (P <0.05). Conclusions:1. Tolerogenic dendritic cells were generated from bone marrow precursors cultured with GM-CSF, VIP, DXM and LPS. The exosomes were then pelleted by ultracentrifugation from the supernatants of tDCs;2. There were similar characteristics between tDex and tDCs in inducting immune tolerance .3. The infusion of tDex in mice with immune-mediated AA can induce CD4+CD25+ Foxp3+ regulatory T cells, let the ratios of Th1/Th2 helper T cells and T-bet/GATA-3 gene expression low and promote hemopoiesis recovery of AA mice.