| Objective:Myasthenia gravis(MG)is an T cell and B cell dependent,complements involved autoimmune disease,caused by acetylcholine receptor antibody.Experimental autoimmune myasthenia gravis(EAMG)is the most ideal animal model of human myasthenia gravis.Dendritic cells(DCs)are antigen presenting cells and can activate native CD4~+T cells.Dendritic cells are divided into mature DCs and immature DCs.Immature DCs can induce immune tolerance or delay the immune response.Tacrolimus,which is an immunosuppressor,can be used to induce immune rejection after organ transplantation and can affect the maturation status of dendritic cells.RelB is a transcriptional regulator.When we knockout the RelB of a mice,it will lack dendritic cell structures in the germinal center,marginal and medullary regions of the spleen and thymus.The main purpose of this study:Construct EAMG using the synthetic peptide of AChR from rat.In vitro,FK506 was used to induce imDCs from rat marrow cells,and then treated EAMG with imDCs.Explore the correlation between MG and the expression of RelB.We hope it can provide a new way for future therapy and diagnosis of myasthenia gravis.Methods:1.Preparation and Identification of Tolerant Dendritic Cells:Cell suspensions were prepared from rat bone marrow and co-cultured with Fk506 at a final concentration of 0ng/ml,5ng/ml,10ng/ml and 20ng/ml respectively.After 6 days Flow cytometry was used to detect the expression of costimulatory molecules CD80,CD86and MHC-II on the surface of dendritic cells.The remaining cells were cultured at the tenth day,and some of the cells were added with LPS 18 hours before harvest for flow cytometry.2.Construction of EAMG:50ul of complete antigen was subcutaneously injected into both sides of the back and shoulder of the Lewis rats at a total amount of 200ul(PBS 100ul,TB 1mg,CFA 100ul,Ag 100ug)and recorded as 0 day.On day 9 and day26,the rats were boosted twice or thrice with a total of 200ul(PBS 100ul,IFA 100ul,Ag100ug)at the same dose and the same place after the first immunization.Rats were weighed daily or every secend day.The clinical symptoms of the rats were observed,and the clinical scores were calculated by measuring muscular weakness.3.Animal groups:The animals were divided into four groups:DCs group modified by tacrolimus,DCs group without tacrolimus modification,tacrolimus group and PBS control group.Corresponding DCs(2×10~6cells/rat)were injected intraperitoneally on day 8 and 17 after the first immunization.The rats in PBS control group were injected with PBS at the dose of 200ul.The rats in tacrolimus group were injected with Fk506 1mg/kg every day or every secend day from the tenth day after the first immunization.Each group of the rats was observed and weighed every secend day,and the clinical symptoms were recorded.4.Preparation of lymph node cells suspension:Remove the lymph nodes at the abdominal aorta,submandibular and axillary under aseptic conditions.Cells suspension were collected and counted after grinding.5.Cell proliferation assay:Lymph node cells containing 4×10~5 cells were cultured in 96-well plates in presence of R97-116 or PBS.All the cells were added with 10ul CCK-8 after 40h of incubation.Then the cells were cultured for 4h at 37℃.At last the OD value was read at 450nm.6.Detection of cell cycle:Lymphocyte suspension were cultured in 24-well plates in presence of R97-116 or PBS for 40h or 60h.The cells were fixed with pre-cooled ethanol at-20℃overnight.Add PI to the cells after washed for 30min at 4℃in the dark.Then the cells were detected by flow cytometry.7.Flow cytometry was used to detect the expression of co-stimulatory molecules on the surface of DCs in peripheral blood of rats:300ul of peripheral blood of each group was collected.The blood was added with fluorescently labeled antibody for30min in the dark.After lysing erythrocytes and being washed,the cells were analyzed by flow cytometry.8.Detection of R97-116 antibody in peripheral blood of rats by ELISA:Plasma was collected from each group rats.Then the level of R97-116 antibody was detected by ELISA.9.The level of RelB m RNA in peripheral blood of EAMG rats and MG patients was detected by RT-PCR.Results:1.The results of flow cytometry showed that the expression of costimulatory molecules CD80,CD86 and MHC-II on the surface of DCs were the lowest incubated with Fk506 at a concentration of 10ng/ml(P<0.05).When the cells were cultured for 6days,the expression of costimulatory molecules on the surface of DCs was lower compared with 10 days.There were significant differences between them.2.The level of AChR antibody in peripheral blood of mice immunized with antigen was higher than that in normal control group.The levels of antibody in im DCs treated group and Fk506 treated group were lower than those in PBS control group and mDCs treated group(P<0.05).The clinical scores of imDCs-treated group and Fk506-treated group were significantly lower than those in the other two groups,which has a significant difference from day 22.3.The results of lymphocyte proliferation showed that the OD value of imDCs-treated group and Fk506-treated group were significantly lower than those of PBS control group and mDCs treated group in presence of antigen peptide.The results of cell cycle showed that the number of S and G2/M phase cells in imDCs treated group and Fk506 treated group were less than those in other groups after 40h and 60h incubation(P<0.05).4.The results of DCs in rat peripheral blood showed that the expression of CD86molecules on the surface of DCs in peripheral blood were significantly different among imDCs-treated group and other groups.5.RT-PCR results showed that the expression of RelB mRNA in peripheral blood of EAMG group was significantly higher than that of normal control group;the expression of Rel B mRNA in peripheral blood of im DCs-treated group and Fk506-treated group decrease compared with PBS control group and mDCs-treated group.In addition,compared with normal people the expression of RelB mRNA in peripheral blood was up-regulated for clinical MG patients,which has a significant difference between them.Conclusions:1.Fk506 can induce tolerogenic DCs in vitro.And the optimal inducing concentration of Fk506 is 10ng/ml.Tolerogenic DCs can be harvested after cultured for6 days.2.The synthetic R97-116 peptide of mouse can successfully induce rat EAMG.3.ImDCs and Fk506 can reduce the clinical scores,increase the body weight and relieve the clinical symptoms of rat EAMG,.4.That mDCs and Fk506 can induce immune tolerance of EAMG rats may have an intensely relationship with decreased serum anti-acetylcholine receptor antibody,inhibited lymphocyte proliferation and down-regulated the expression of RelB mRNA.5.The expression of RelB mRNA in peripheral blood of EAMG rats and clinical MG patients increased,indicating that the occurrence of myasthenia gravis was closely associated with the expression of Rel B. |
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