The Preliminary Therapeutic Effect On Mice Experimental Autoimmune Encephalomyelitis Of Rat Bone Marrow Mesenchymal Stem Cells Transplantation

Objective: To investigate the therapeutic effect of allogeneic transplantation of rat bone marrow mesenchymal stem cells(BM-MSCs)on experimental autoimmune encephalomylitis(EAE)in mice.Methods: 1.Rat BM-MSCs were obtained by whole-bone marrow adherent culture method,observed for cell morphology,Identified with flow cytometry for P3 cells surface antigen markers CD29,CD90,CD106,CD45.Then the obtained Rat BM-MSCs were induced osteogenic and adipogenic differentiation.2.Twenty female C57BL/6 mice were randomly divided into three groups:normal control group,PBS group and Rat BM-MSCs group.Rat BM-MSCs were transplanted into Rat BM-MSCs group,PBS mice were transplanted as PBS group,and untreated mice were used as normal control group.MOG35-55 combined with complete Freund's adjuvant after full emulsification,then immunization by subcutaneous multi-point injection to induce EAE.3.The mouse were treated with Rat BM-MSCs(1×106cells/200?l)by intraperitoneal injection 38 days after immunization,the rats in the PBS group were treated with the same amount of PBS,after 10 days of re-treatment,Neurological function scores were performed to evaluate the neurological function everyday.4.12 days after the second treatment,the spinal cord of each group was treated with HE staining and Luxol Fast Blue staining to observe the inflammatory cell infiltration and myelination of spinal cord.5.On the 12 th day after the second BM-MSCs transplantation,take the spleen of each group to be single-cell suspension.The proliferation of splenocytes was detected by flow cytometry after stimulation with 10?g/ml ConA and MOG35-55 for 3-5 days after splenocytes CFSE labeling.6.Mouse peripheral blood ser?m was collected on the 12 th day after the second passage of Rat BM-MSCs.The contents of cytokines(IFN-??IL-17)in peripheral blood of normal control group,PBS group and Rat BM-MSCs group were evaluated by ELISA.Results: 1.Rat BM-MSCs were successfully isolated and cultured.The third generation cells were spindle-shaped or polygonal,showing typical spiral growth.Flow cytometry results showed that P3 cells of Rat BM-MSCs were expressed CD29,CD90 and CD106,not expressed CD45,and the obtained cells could be induced to osteogenic cells and adipogenic cells.2.In the C57BL/6 mice,the neurological function of EAE mice was aggravated and the clinical score was increased.After BM-MSCs transplantation,the neurological deficits and the clinical score of Rat BM-MSCs group were less than those in PBS group,Clinical symptoms remained further improved after the second transplantation.3.HE staining indicated that the infiltration of inflammatory cells in Rat BM-MSCs group was significantly lower that in PBS group(P <0.05).Luxol Fast Blue staining displayed that the demyelination of Rat BM-MSCs group was less than the PBS group at the same time point(P <0.05).Pathological analysis showed that the infiltration and demyelination of spinal cord were improved after BM-MSCs transplantation.4.Flow cytometry analysis showed that the proliferation of splenocytes in PBS and Rat BM-MSCs was increased after ConA and MOG35-55 stimulation,while that in Rat BM-MSCs group was lower than the PBS group.5.Compared with the normal control group,the levels of proinflammatory cytokines IFN-? and IL-17 in the peripheral blood of PBS group were significantly higher than those in the control group(P <0.05).Compared with the PBS group,the levels of proinflammatory cytokines IFN-? and IL-17 in blood plasma were decreased(P<0.05).Conclusion: Rat BM-MSCs can be effectively isolated and purified by whole bone marrow adherent culture method,and rat bone marrow mesenchymal stem cells have therapeutic effect on mouse EAE.