Study On Immunoreaction Type In Different Course Of Disease In Experimental Autoimmune Encephalomyelitis

Background:Multiple sclerosis is considered to be an autoimmune disease mediated by CD4~+Th1 cells,which is the dominant theory that Th1 mediated cell immunity in the study.In recent years,studies have proved that Th17 cells play a critical role in the central nervous demyelinating autoimmune disease,that imbalance between Th17 and regulatory T cells may also be an important cause of disease pathogenesis mechanism.In addition,studies have shown that humoral immunity also plays an important role in the pathogenesis of MS.The mechanism of B cells in MS pathogenesis is still not very clear.The intrathecal IgG synthesis in MS patients indicated specific antibody production.In the pathologic examination,the lesion had the axis cylinder degeneration besides myelinoclasis.Myelin and axon protein may be the target antigen of autoantibody attack.Therefore,the search for antibodies related to neure and axis cylinder degeneration can both confirm the role of humoral immunity in MS pathogenesis,and also find a biological indicator conducive to MS diagnosis.Objective:Experimental autoimmune encephalomyelitis(EAE)mouse model is established and evaluated;To explore the changes of subgroups of T cells and B cells during the onset,peak and remission of the course of disease;To explore the changes of cytokines at different stages in EAE mice.Explore the levels of triose phosphate isomerase(TPI)-reactive antibody and glyceraldehyde 3 phosphoric acid dehydrogenase(GAPDH)-reactive antibody in the expression of EAE mice,which can look for potential target antigen.To explore immunoreaction type in different course of disease and mutual influence of immune response.Method:C57BL/6 mice were randomly divided into:EAE group(n=18),which was prepared by using myelin oligodendrocyte glycoprotein(MOG)35-55peptides/complete Freund’s adjuvant(CFA);The control group(n=18)treated the mice with normal saline.The peripheral blood T cell subsets(Th1,Th2,Th17,Treg,gamma delta T cells)and B cells(initial mature B cells,memory B cells and plasma cells)were detected by flow cytometry on immune 0,3 days,7 days,14 days,21days,30 days.The brain and spinal cord were taken out for HE staining and LFB staining on 7 days after immunization(early),18 days after immunization(peak),30 days after immunization(remission).Where the T cell and B cell subsets related proteins located were detected by immunohistochemical staining at the same time.During the same period,tissue homogenation was taken and detected the changes of T cells and B cells related cytokines and TPI and GAPDH antibodies by ELISA in the brain and spinal cord tissues of rats.To analyze the types of immune responses and their influence on the different stages of neurological dysfunction in EAE mice.Results:1.The EAE animal model was successfully established by using MOG35-55peptides/CFA to induce C57BL/6 mice.The mice in the EAE group showed clinical symptoms,the average weight was lower than the control group,and the neurological function score was higher than the control group,with statistical difference(P<0.05).A large number of inflammatory cells were observed in the brain and spinal cord tissues of EAE mice in HE staining.A large patch of the balloon samples area was found,which was depigmentation area for LFB not shaded,in the brain and spinal cord tissues of EAE mice in LFB staining.It meansdemyelination2.Flow cytometry test results show that Th1 cells in the control group have no obvious change from 0 day until 31 days,EAE group began to drop compared to the baseline on 14 day and is lower than the control group on 14,21,30 day,there are significant differences(P<0.05).Th2 cells in the control group have no obvious change from 0 day until 31 days,EAE group began to rise compared with the baseline on 14 day and is higher than the control group on 3,7,14,21 day,with significant difference(P<0.05).Th17 cells in the control group have no obvious change from 0 day until 31 days,EAE group began to rise on 3 day compared with the baseline and is higher than the control group on 3,7,14,21 day,with significant difference(P<0.05).The two groups of Treg cells were both low expressed and have no statistical difference.Compared with the baseline,there was a statistically significant difference compared with the control group in subset ofγδT cell(P<0.05).Immunohistochemical staining showed that the protein expression level related T cells was significantly higher than that of control group in spinal cord tissues of EAE group.It was also higer in the brain tissue of EAE group but was not match for the spines.IL-10,IL-23,TNF-?in EAE group were higer than control group on 7 day,IL-10,IL-17,IL-23 were also higher than control group on 18 day,both with significant difference(P<0.05)..In addition,IL-10,IL-17,IL-23 were increased in EAE group on 18 day compare with 7 day,and IL-23 was decreased on 30 day compare with 18 day,both with significant difference(P<0.05).3.Flow cytometry test results show that the initial mature B cells began to decrease in the EAE group on 7 day,and maintained to 30 days,with a statistically significant difference from baseline(P<0.05).It was lower than the control group on 14,21 and 30 days(P<0.05).Memory B cells in EAE group were both lower than control group.There was a statistically significant difference that it increased on3,7,14 and 21 days compared with the baseline(P<0.05).There was a statistically significant difference in plasma cells on 14 and 21 days,compared with the baseline,and statistically significant difference was observed on 14 day compared with the control groups(P<0.05).Immunohistochemical staining showed that the protein expression level related B cells was significantly higher than that of control group in spinal cord tissues of EAE group.It was not different between the brain with the spines.IL-10,IL-12,TNF-?in EAE group were higher than control group on 7 day,IL-10 was also higher than control group on 18 day,both with significant difference(P<0.05).In addition,IL-10 was increased in EAE group on 18 day compare with7 day,with significant difference(P<0.05).4.TPI,GAPDH in EAE group were higher than control group on 7 day,but both decreased on 18 day,both with significant difference(P<0.05).Conclusion:By using the method of MOG35-55/CFA,C57BL/6 mice can be successfully established the EAE animal model.The operation is simple and reliable,and the model has good stability and repeatability.Th17 cells increased in the initial course of disease and maintain a high level in EAE mice.It prompt Th17 cells is the key cell which mediated diseases,and perform the whole course.As protective T cells,Treg decreased significantly,suggesting that the imbalance of Th17/Treg was the cause of disease occurrence and progression.B lymphocytes are involved in the process,and all kinds of B cells are expressed in order of differentiation,and not only reacted by antibodies.The secretion of pathogenic cytokines is the main mechanism.GAPDH and TPI antibodies have been expressed in the early stage of the disease,which may be one of the causes of neuronal and axonal injury,but not the pathogenic antibodies that penetrate the entire course of the disease.