The Establishment Of Murine Experimental Autoimmune Encephalomyelitis Model And The Study Of The Role Of NKT Cell And Th1/Th2 Cell In EAE

Objective Multiple sclerosis is an inflammatory and demyelinating disease of the central nervous system ,which is characterized by the relapsing,deferment and high disability.Also individual genetic susceptibility and environmental factors contribute to autoimmune disease occur. Because the sample of MS is not easy to be obtained and exact pathogenesis remains unclear,it is necessary to establish animal model to study MS.In this study,to establish stable and reliable animal model with high incidence,we induced experimental autoimmune encephalomyelitis(EAE) models of C57BL/6 mice by myelin oligodendrocyte glycoprotein,MOG35-55,and explore the immunologic mechanisms causing EAE,it is supposed to provide basis for the pathogenesis,prevention and therapy of MS.Methods1. Each mouse was immunized subcutaneously with 300ug of MOG35-55 emulsified in CFA containing 1.0mg H37Ra and then immediately injected intraperitoneally with pertussis bacterium and again 2 days later.The clinical symptoms,weigtht and hisopathologic changes of the central nervous system(CNS) were observed.2. Use the SYBR fluorescent quantitation polymerase chain reaction technology to detect the interferon-γmRNA and interleukin-10mRNA expression in mouse nervous tissue(brain tissue and myeloid tissue);Use the Flow Cytometry(FCM)technology to measure CD1d,B7H1,B7H2 expression on the mouse splenic lymphocyte .By comparing the model group and control group diffreences,we explore the immunologic mechanisms causing EAE.Results1. The EAE group developed the typical symptoms of EAE with the incidence of 65%,the highest clinical score is up to 5.The infiltration of inflammatory cells in brains and spinal cords,biopsy showed dense perivascular cuff-like emergence inflammatory cells.Inflitration with lymphocyte predominant cell type,can also be found a small amount of glial cell line.Gray matter are involved,white matter nerve fiber bundles,the meninges and the meningeal infiltration also found inflammatory cell.2. Compared to the normol contro l group△CT(12.70+0.16),in the model mice,real-time quantitative PCR results showed that the target gene IFN-γmRNA decreased to(10.89+0.52), P<0.05; Compared to the normol contro l group△CT(17.36+1.01),in the model mice,real-time quantitative PCR results showed that the target gene IL-10mRNA step up to(19.12+0.61), P<0.05.Whears there was no significant difference about the expression of IL-18mRNA between model group and control group.3. Flow cytometry results showed that:compared to the control group mouse spleen cell suspension CD1d molecules'expression on APC mean percentage of cells(9.32+1.07)%,the percentage of mean peak incidence in model group is (2.09+0.26)%,the model group in remission is (13.8+1.93)%,comparing the differences between the groups were statistically significant(P<0.05). Compared to the control group mouse spleen cell suspension B7H1 molecules'expression on APC mean percentage of cells (55.01+3.12)%,the percentage in model group is (68.23+4.84)%,it showed significantly higher(P<0.05).Compared to the control group mouse spleen cell suspension B7H2 molecules'expression on APC mean percentage of cells(22.59+2.87)%,the percentage in model group is (30.21+3.37)%,it showed significantly higher(P<0.05).Conclusion1. We will fully emulsified MOG35-55 with CFA,were subcutaneously immunized C57BL/6 mice,plus intraperitoneal injection of pertussis bacilli,a successful construction of the C57BL/6 chronic monophasic EAE model,which is not only the high incidence,stable and reliable,and the pathological changes close to MS.It is an ideal animal model for MS research. 2. By comparing the EAE mice andnormal mice,Th1/Th2 cells in inflammatory cytokines(IL-18, IL-10, IFN-γ) gene and protein level, and B7-H1, B7-H2 membrane molecule expression of splenic lymphocytes of different from, display, T-cell activation,cytokine has led to a possible co-occurrence of EAE. The expression of CD1d suggests NKT cells in EAE disease in remission period of protection. For the future further work on the pathogenesis and treatment of MS research foundation.