| Objective:To investigate the feasibility of IL-35gene transfection of mice bone marrow mesenchymal stem cells (MSCs) and the effect of IL-35gene modified MSCs on the immune function of mice.Methods:(1) Harvest the Balb/c mice bone marrow from the tibia and femur, culture mesenchymal stem cells in vitro. Adipogenic differentiation was induced with dexamethasone, insulin, indomethacin, and IBMX. Osteogic differentiation was induced with dexamethasone, β-glycerophosphate and vitamin C. CD105, CD90, CD44, CD45, CD34and SCA-1on the mesenchymal stem cells are examined by the flow cytometry (FCM). Adipogenic and osteogenic cells were stained with oil red O and alizarin red.(2) IL-35gene expression plasmid and the green fluorescent protein expression plasmid (L6) were transfected into MSCs in vitro by cationic liposome transfection.Transfection efficiency was evaluated by FCM. IL-35concentration in the supernatant after cell transfection was determined by enzyme linked immuno sorbent assay (ELISA).(3) IL-35modified MSCs were injected into BALB/c mice through tail vein. The concentration of IL-35in serum after the cell treatment was determined by ELISA. The proportion of CD3+cells, CD4+T cells, CD8+T cells and CD4+CD25+Treg in mice peripheral blood was analyzed by FCM.(4) Set up one-way mixed lymphocyte culture with the spleen mononuclear cells which was isolated from BALB/c mice and C57BL/6mice. The impact of IL-35modified MSCs on cell proliferation and the proportion of CD3+cells, CD4+T cells, CD8+T cells and CD4+CD25+Treg were determined.Results:(1) The mice bone marrow MSCs can be isolated and cultured by the whole bone marrow adherent method. The cell morphology after isolation and amplification was elongated spindle, vortex-like. The MSCs showed positive expression of CD105, CD90, CD44and SCA-1, and negative expression of CD45and CD34;3rd generation of mesenchymal stem cells were induced to adipogenic cells and osteoblasts.(2)Green fluorescent protein expression can be observed in L6transfected MSCs. Thetransfection efficiency is about27.40%. IL-35is expressed in the MSCs supernatant after IL-35transfection.(3)After the injection of IL-35modified MSCs to mice, IL-35was detected in the serum; Mice treated with IL-35 modified MSCs showed increased percentage of CD4+CD25+Treg(5.24±0.61%) in peripheral blood. There is the statistical significance compared with other groups (P <0.05). A reduction of CD4+T cells percentage was seen compared with the saline group.(4) IL-35modified MSCs up-regulated the CD4+CD25+Treg(4.35±0.57%)level in the allogeneic mixed lymphocyte reaction system, lowered the percentage of CD3+cells(29.65±4.44%) and the CD4+T cells(14.47±3.86%)level,(P<0.05).Conclusion:The mice bone marrow mesenchymal stem cells can be isolated and cultured by the whole bone marrow adherent method. Mesenchymal stem cells have multilineage differentiation potential, and its can be differentiated into adipocytes and osteoblastsand. positive expression of CD105, of CD90, of CD44and the SCA-1, IL-35transfected MSCs can induce proliferation and differentiation of mice CD4+CD25+Treg, and directly or indirectly inhibit the activation level of CD3+cells,CD4+T cells, and thus get a favoriateimpact on the establishment of immune tolerance during transplantation. |
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