Therapeutic Effects And Mechanism Study Of Menstrual Blood-derived Mesenchymal Stem Cell On Experimental Autoimmune Encephalomyelitis

BackgroundMultiple Sclerosis（MS）is an autoimmune disease of the central nervous system,in which immune system attacks the myelin sheath surrounding the axon,in cause of demyelination and nidus.The experimental autoimmune encephalomyelitis（EAE）,an animal model,has similar pathological characteristics with MS,so EAE has become an important platform for the research and treatment of MS.Menstrual blood-derived stem cells（MenSC）are superior to other sources of mesenchymal stem cells,such as bone marrow,umbilical cord,fat because of their non-aggressive,short-cycle and repeatability.To now,there are no reports about the use of MenSC to treat EAE.On the other hand,although there are many reports on the treatment of EAE with stem cells,the mechanism is unclear yet.Therefore,this subject aims to explore the mechanism of action of MenSC on EAE,while exploring the therapeutic effect of MenSC on EAE.Objects1.Establish a culture system of MenSC.2.Establish the mouse EAE model.3.To explore the effect that MenSC treat EAE.4.To explore the mechanism that MenSC treat EAE.Methods1.Isolate,culture and identify the mesenchymal stem cells getting from menstrual blood to provide high quality and enough quantity cells for subsequent experiments.2.Establish mouse EAE model by injecting MOG35-55 mixed with complete Freund’s adjuvant containing heat-killed mycobacterium by subcutaneous,and injecting of the same dose of PTX on the same day with injecting MOG_35-55 and also on the second day by tail vein.3.Intravenous injection（i.v.）,intraperitoneal injection（i.p.）,animal anatomy,extraction of individual cells from various organs,cell surface staining,intracellular staining and flow cytometry provide experimental technical basis for cell injection,vivo and vitro animal experiments and mechanism exploration respectively.Real time PCR was used to detect the expression of various genes of MenSC.Results1.Mesenchymal stem cells extracted from menstrual blood have been identified and demonstrated to meet the certification standards for international mesenchymal stem cells.】2.Inject 1×10⁶ MenSC on one day（day-1）before EAE induction,the sixth day（day6）,the tenth day（day10）,nineteenth day（day19）after EAE induction respectively.The results showed that MenSC injected on day-1,day6 and day10 all can inhibit the progression of EAE disease,the effect of MenSC injected on the day6 is most significant,but the injection of stem cells on day19 did not ameliorate the clinical score of EAE.3.By comparision the two ways to inject MenSC by i.p.and i.v.,both of them can decrease theclinical score of EAE,but the inhibition effect by i.v.is better than i.p.4.The percentage and numbers of CD4⁺T cells,Th1,Th17,MOG-reactive Th1 and Th17 cells of the peripheral immune organs and CNS from EAE mice treated with MenSC were lower than the EAE mice untreated with MenSC,especially in spleen.Conclusions1.Injection of MenSC on day-1,day6 and day10 all can ameliorate the clinical symptoms of EAE,and injection on day6 exhibit the best therapeutic effect.However,the clinical score of EAE could not be reduced by injecting MenSC on day19.2.MenSC may play a role in EAE mice by inhibiting CD4⁺T cells,Th1,Th17,MOG-reactive Th1 and Th17 cells in peripheral immune organs and central nervous system.