| Backgroud and objective Mesenchymal stem cells(MSCs), a rare population of non-hematopoietic stromal cells, are able to adhere to plastic and form fibroblast colonies, with self-replicating, self-renewal and multi-differentiation potential, existing in the bone marrow and massive connective tissues of body, which are the important member of stem cells. MSCs are multipotent cells even after continuous passage culture and cryopreservation able to differentiate into adipocytes, chondrocyte, osteocytes, myocytes, tenocytes, neurocytes, hepatocytes, cardiocytes, islet β cells, endotheliocy tes and so on. The expressions of MSC have obvious features: high expression of CD90, CD105, CD44, CD73, CD4 molecules etc, and without the expression of hematopoietic cell surface molecules CD34, CD45, CD11 b, CD11 c, CD14, CD19, CD86 and MHC-Ⅱ. Therefore, we can estimate the purity of MSCs after in vitro cultivation and purification according to their characteristics, which can guide the study of MSCs. Moreover, no matter autologous nor allogeneic MSCs will not lead to immune responses in the host, that is the low immunogenicity of MSCs, which make it possible to be applied to the study of cell therapy. In recent years, the statement that MSCs inhibit the activation and proliferation of allogeneic Tlymphocytes was confirmed. Some say that MSCs can exert their immunosu ppression through releasing soluble cytokines; other studies have found that MSCs possessed the function of immunoregulation by cell-cell immediate contact with target cells; even some scholars say that the immunoregulation of MSCs was accompanied with the change of cell apoptosis and cell cycle. Along with the study enthusiasm that MSCs are applied in treating autoimmune disease or tumour etc, more people start to pay attention to their clinical effects and time duration. Consequently, the objective of this study is to observe the curative effect when MSCs possessing green fluorescence GFP are transplanted to the C57BL/6 mice model of multiple sclerosis-experimental autoimmune encephalomyelitis, and discuss the mechanism of immunoregulation from the cytokines and cellular level, and tracking the action sites, number and action time of MSCs with GFP flurescence labeling in recipient C57BL/6 mice, for knowing about the durability of therapeutic effect.Materials and methods 1. male C57BL/6 and GFP-C57BL/6 mice [VITAL RIVER company, Animal permit number: SCXK(Beijing) 2012-0001] are 20-25 g and 6-8-week old considered as the subjects of the experiment. 30 C57BL/6 mice were induced to EAE mice considered as recipient mice, and 20 GFP-C57BL/6 mice considered as donor mice were executed and achieved their GFP-MSC cells. The MSC treated group was transplanted with GFP-MSC cells in vein, the EAE group was transplanted with equal PBS, and the normal group was untreated.2. the models of EAE were established: 5mg/ml CFA and 1.5mg/ml MOG35-55 were isovolumetric 1:1 mixed. The water-in-oil emulsion was formed by pushing and pulling over and over again. C57BL/6 mice were immunized with the water-in-oil emulsion by 200ul/mice in subcutaneous. Pertussis toxin(PT) was injected at 0 and 2 days after MOG immunization into caudal vein 2ul/mice(diluted in 200 ul PBS).3. obtaining GFP-MSC cells: GFP-C57BL/6 mice were executed by cervical dislocation. The tibia and femur were peeled to expose their marrow cavity and the bone marrow cells were washed from skeletons by DMEM culture solution with 15% FBS under aseptic conditions, and cultured in 37℃ 35% CO2 incubator. Non-attached cells were removed by exchanging culture solution, which was operated after 4 hours, after which the exchanging frequency was changing to 12 hours. Then, these cells were passaged once every 3 days or so according to the rate of covering cells(pancreatin digests attached cells, and then continue culturing in new culture solution). In our study, we used light microscopy and flow cytometry to identify their purity.4. obtaining the spinal cord sections of the EAE group and MSC-treated group mice after cell-transplanted 15 days and staining HE: The mice were anesthesiaed by 100 ul 20g/L pentobarbital sodium; the mice were experienced cardiac perfusion with 40g/L paraformaldehyde. The spinal cord tissues were achieved, and then were fixed in paraformaldehyde about 24 hours. The treated tissues were sent to the pathology department of the first affiliated hospital of Zhengzhou university to slice tissue section and stain HE.5. the contents of cytokines(TNF-α, IFN-γ, IL-4 and TGF-β) in peripheral blood of the MSC treated group, EAE group and normal group mice were detected by ELISA methods, and the acquiring data were analyzed by statistics6. the percent of Treg cells in splenic cells in recipient mice after 2 and 10 days MSCs transplantation was measured by flow cytometry. At the same time, the percent of GFP+ cells in the mononuclear cells of spinal cord was tested at those two points.Results 1. The cultivation and purification of mesenchymal stem cells succeeded. MSCs passaged to the 5th generation were observed by optical microscope: the shape of MSCs was clostridial, fusiform or polygon form etc. The results of flow cytometry showed that the expressions of more than 95% MSCs at 5th generation was CD34-CD45-CD105+.2. The effect after transplantation of MSCs to EAE mice was significant: compared with the control mice, the clinical score of MSC-treated mice experienced significantly reduce at 2 days after MSC transplantation(p<0.05), after which the clinical symptoms of EAE mice were improved at a slow rate. The histologically analysis of spinal cord sections showed that the condition of nucleated cell infiltration improved after 15 days transplantation.3. Compared with normal mice, the contents of proinflammatory factor TNF-α(21.94±0.46 pg/ml) and IFN-γ(203.8±0.19 pg/ml) in periphery blood of EAE mice were significantly increased(p < 0.05), and the contents of anti-inflammatory cytokine IL-4(12.87±0.14 pg/ml) and TGF-β(6.74±0.06 pg/ml) were obviously decreased(p<0.05). Compared with EAE mice, the contents of proinflammatory factor TNF-α(13.82±0.24 pg/ml) and IFN-γ(100.0±0.31 pg/ml) in periphery blood of the MSC-treated mice experienced apparent increase(p<0.05), and the contents of anti-inflammatory cytokine IL-4(27.30±0.22 pg/ml) and TGF-β(12.09±0.09 pg/ml) were obviously increased(p < 0.05); even, the contents of IFN-γof MSC-treated mice were lower than normal mice(141.5±0.21 pg/ml)(p<0.05).4. The percent of Treg cells in splenic cells of recipient mice was analyzed by flow cytometry: the percent of Treg cells in splenic cells of MSC-treated mice(19.08±0.33) was obvious higher than the EAE mice(6.32±0.19) and normal mice(11.20±0.37)(p<0.01).5. the percent of GFP+MSC cells in mononuclear cells of spinal cord in recipient mice after 2 days and 10 days MSC transplantation was analyzed by flow cytometry: the percent of GFP+MSC cells was(1.82±0.08)% after 2 days MSC transplantation, and the GFP+MSC cells almost disappeared after 10 days transplantation.Conclusion 1, the curative effect of MSC to EAE mice is obvious; 2, the treatment of MSC to EAE are connected with regulating the secretion of cytokines and upregualating the Tregs; 3, the treatment of MSC to EAE still exist after their disappearing in a certain time. |
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