| Section One The establishment and identification of experimental autoimmunehepatitis in C57BL/6 miceObjective: To establish the animal model of autoimmune hepatitis for the study of disease pathogenesis and effect of novel treatment. Methods: Experimental autoimmune hepatitis(EAH) was established in C57BL/6 mice with freshly prepared syngeneic liver antigen (S-100) and complete Freund's adjuvant(CFA) by twice of peritoneal injection on the first and seventh day. The normal control mice were injected with equal volume of PBS. The mice were then killed at the end of the fourth week to detect the serum levels of ALT and AST, antibodies of ANA and SMA, and histological examination of liver tissue was performed. All results were compared with normal controls. Results: The general condition was poor in pathological mice, and the increasing of weight was not significant (p=0.000). The serum levels of ALT and AST were significantly higher than normal controls (ALT:47.63±7.45 v.sl9.95±0.99, p=0.001; AST:248.37±21.25 v.s.93.00±16.61, p=0.000). The positive rate of ANA was 26.7%, and SMA was not found. The histological examination of liver tissue showed there were lots of inflammatory cells, most of lymphocytes and plasma cells infiltrated in the portal area, and presence of interface hepatitis.Conclusions: C57BL/6 is immunized with syngeneic liver antigen and complete Freund's adjuvant (CFA) by twice of peritoneal injection. On the four weeks after immunization, the laboratory and histological findings are coincident with human AIH, which indicates the animal model of AIH is successfully established.Section Two The expression levels of PD-1, PD-L1, PD-L2 in the liver ofexperimental autoimmune hepatitis established in C57BL/6 miceObjective: To explore the role of PD-1 (programmed death-1) /PD-L1,L2 (programmed death-1 ligand 1,2) system in the pathogenesis of experimental autoimmune hepatitis (EAH). Methods: Experimental autoimmune hepatitis(EAH) was established in C57BL/6 mice and the expression levels of PD-1 and PD-L1,L2 in the murine liver and the cytokines, IFN-γ,TNF-αand IL-4 in the spleen were detected using retrotranscription-PCR(RT-PCR), and results were compared with normal controls. Results: The expression levels of PD-1,PD-L1,PD-L2 mRNA were higher in EAH compared with normal controls(p<0.05), the PD-L2/PD-1 ratio was relatively lower in EAH (EAH -0.08±0.35, normal controls 0.52±0.07, p=0.009). In the EAH, the expression of 3 cytokines were all upregulated compared with normal controls. PD-L1 had a positive correlation with the expression of IFN-γ(r=0.289, p<0.05), while PD-L2 showed a positive correlation with both expressions of IL-4 (r=0.378, p <0.01) and IFN-γ(r=0.261, p<0.05). The ratio of PD-L1 to IFN-γand PD-L2 to IFN-γ, especially the ratio of PD-L2 to IFN-γ, were lower in EAH compared with normal controls. While TNF-αshowed no correlation with PD-L1 (r=0.044, p=0.736) or PD-L2 (r=0.127, p=0.335). Conclusions: The expression of PD-1/ PD-L1, L2 is upregulated in EAH and regulated by cytokines of IFN-γand IL-4. PD-1 system maybe play an important role in the pathogenesis of AIH.Secton Three The application of tumor necrosis factor a antagonist on theC57BL/6 mice with experimental autoimmune hepatitisObjective: To investigate the effect of tumor necrosis factor a antagonist on the experimental autoimmune hepatitis (EAH) established in C57BL/6 mice. Methods: Experimental autoimmune hepatitis(EAH) was established in C57BL/6 mice, and tumor necrosis factor a antagonist was used in C57BL/6 mice with the dose of 0.1mg/0.2mg, twice a week for four weeks at the end of the second and fourth week after the first immunization, then the serum levels of ALT and AST were detected, histological examination was carried out, and RT-PCR was performed to determine the expression levels of TNF-α, TRAF-1 and TRAF-2. All results were compared with pathological controls and normal controls. Results: After 4 weeks of using tumor necrosis factor a antagonist, the serum level of liver transaminase decreased, but was not significant statistically. Histological findings of liver tissue showed liver inflammation improved slightly in treatment group, and there was no aggravation after using antagonist. The expression level of TNF-αwas decreased significantly after treatment (p=0.019). Compared with normal controls, the expression level of TRAF-1 was increased (p=0.422), while TRAF-2 was decreased significantly (p=0.007); after treatment with antagonist, the expression levels of TRAF-1 and TRAF-2 were both upregulated (p=0.422 and p=0.015, respectively). The expression of TRAF-2 was correlated with TNF-α(r=0.261, p=0.044). Conclusions: TNF-α, TRAF-1 and TRAF-2 are involved in the inflammatory reaction of liver in AIH, which indicates TNF-αis a new target for the treatment of AIH and tumor necrosis factor a antagonist may be used clinically in the future.
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