| Objective To investigate the effects of apolipoprotein E(ApoE)on the expression of tumor necrosis factor ?(TNF-?),interleukin(IL-17)and Matrix-9(MMP-9)in spleen lymphocytes of mice immunized with myelin antigen.Method Dilutions of oligodendrocyte glycoprotein(MOG35-55)to 2 mg/ml with 0.01 mol/L PBS buffer solution,with equal amounts of complete Freund's adjuvant containing 4 mg/ml heat-killed tuberculin(H37Ra).The agent is thoroughly mixed and a water-in-oil emulsion antigen is prepared by whipping on ice using a full glass syringe.The prepared MOG35-55 emulsion antigen(0.2ml/mice)was injected subcutaneously in the dorsal subcutaneous C57BL/6J mice of the homologous ApoE-deleted(ApoE-/-)and wild-type(WT)types.On the day of immunization and 48 hours after immunization,each mouse was injected intraperitoneally with pertussis toxin(200 ng/time/mice).At 7 days after the induction of immunity,the spleens of mice were harvested,and lymphocytes were isolated and extracted.In vitro culture was performed.The lymphocytes extracted from the two mice were randomly divided into four groups: ApoE-/-experimental group(E-ApoE-/-),WT experiment group(E-WT),ApoE-/-control group(C-ApoE-/-),WT control group(C-WT).Lymphocytes of two experimental groups(E-ApoE-/-group and E-WT group)were stimulated with 25 ?g/ml of MOG35-55,and two control group lymphocytes(C-ApoE-/-group and C-WT group)were given An equal amount of physiological saline was used for treatment.After 24 hours,each group of lymphocyte culture supernatants was centrifuged and collected.Enzyme linked immunosorbent assay(ELISA)was used to detect the concentrations of tumor necrosis factor alpha(TNF-alpha),interleukin-17(IL-17),matrix metalloproteinase-9(MMP-9)and tissue inhibitor of metalloproteinase-1(TIMP-1)in each group and carry out statistical analysis.Result(1)After the induction of myelin antigen in ApoE-/-mice and WT mice,their splenic lymphocytes(C-ApoE-/-group and C-WT group)can secrete TNF-?,IL-17 and MMP-9.The concentrations of TNF-?,IL-17 and MMP-9 in spleen lymphocytes of ApoE-/-mice(C-ApoE-/-group)were higher than those in WT mice(C-WT group),and the difference was statistically significant(P<0.05).(2)In vitro,two mice spleen lymphocytes were stimulated by myelin antigen MOG35-55,and the concentrations of TNF-?,IL-17 and MMP-9 in lymphocytes of two experimental group(E-ApoE-/-group and E-WT group)were significantly higher than those of two control group(C-ApoE-/-group and C-WT group).The concentration of E-ApoE-/-group was more significantly elevated than the E-WT group,and the difference was statistically significant(P<0.05).(3)Immune induction and in vitro use of MOG35-55 to stimulate the spleen lymphocyte of mice,all of which were able to express TIMP-1,but the TIMP-1 concentrations of splenic lymphocytes in each group were not significantly different,and no significant difference was found(p>0.05).Conclusion After myelin antigen stimulated mouse splenic lymphocytes,lymphocytes could express inflammatory cytokines TNF-?,IL-17,matrix metalloproteinase-9(MMP-9)and matrix metalloproteinase inhibitor-1(TIMP-1).Apo E deficiency can significantly increase the secretion of inflammatory factors TNF-a,IL-17 and matrix metalloproteinase-9(MMP-9)in the spleen lymphocyte,which may affect the integrity of the blood brain barrier,but ApoE deficiency has no significant effect on the TIMP-1 expression in the spleen lymphocytes of mice.The relationship between Apo E,inflammatory factors,and matrix metalloproteinase-9 may serve as the starting point for the study of blood brain barrier damage mechanism and its therapeutic strategy in immune mediated central nervous system inflammatory demyelinating diseases,such as multiple sclerosis. |
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