| Objective To overexpress Galectin-9in the acute rejection models of rat liver transplantation to explore the possible role of Galectin-9in the formation of induced immunological tolerance in acute rejection models of liver transplantation.Methods (1) The acute rejection models of rat orthotopic liver transplantation were established by modified Kamada "Two-Cuff "methods, The Lewis rats were used as donors, The BN rats were used as recipients. The recipients were divided into three groups according to the principle of completely randomized controlled trials. The transfected group (n=10), the blank plasmid group (n=10), control group (n=10) respectively. In transfected group, clamping the suprahepatic vena cave, hepatic inferior vena cava and portal vein of liver graft by the use of noninvasive vascular folder respectively during the clod ischemia period,2ml recombinant Ad-galectin-9(1×109IU/ml) was injected through the portal vein, and then preserves it for sixty minutes. In blank plasmid group,2ml adenoviral vector (1×109IU/ml) was injected through the portal vein by the same approach, also preserve for sixty minutes. In controlled group,2ml0.9%saline were injected through the portal vein; also have the sixty minutes preservation.(2) Five recipients in each groups were sacrificed on the seventh day posttransplantation, the blood from inferior vena cava and the middle lobe of allograft liver were collected for the further examination. The rest rats in each groups were used to observe the post transplantation survival rate.(3) Liver allograft specimens were observed under the optical microscope after hematoxylin-eosin staining. The extents of acute rejection were assayed by pathological examination and were graded by the Banff scheme. Liver functions were evaluated through total bilirubin (TBIL), alanine aminotransf erase (ALT) and aspartase mainotransf erase (AST) with an automatic biochemical analyzer. The relative mRNA expression level of Galectin-9, T-bet and RORyt in allograft liver tissues were detected by Real-time PCR respectively. The Western blot were performed to determined the protein expression level of interferon-y(IFN-γ)及interleukin-17(IL-17) in allograft liver tissues respectively. The plasma level of IFN-y and IL-17were detected by Enzyme-Linked Immunosorbent.Results (1) The acute rejection models of Lewis rat to BN rat orthotopic liver transplantation was successfully established.(2) Survival rate of transfected group is eighty percent, while the survival rate of the other two groups are twenty percent. The survival rate in transfected group is much higher (P<0.05)(3) According to the RAI score criterion, the transfected group show slight acute rejection, the Banff score classified it to Ⅰ-Ⅱ and the blank plasmid group and controlled group display severe acute rejection, and classified to Ⅲ.(4) The plasma level of TBIL in transfected group, blank plasmid group and controlled groups were21.35±0.47μmol/L、89.46±17.42μmol/L、92.35±15.45μmol/L respectively, the plasma level of ALT in transfected group, blank plasmid group and controlled groups were216.47±13.62U/L,912.56±44.21U/L、957.71±32.58U/L respectively, while the plasma level of AST in transfected group, blank plasmid group and controlled groups were136.68±21.32U/L、687.93±64.18U/L、697.37±53.42U/L, the plasma level of TBIL, ALT and AST were significantly lower in transfected group.(P<0.05)(5) The Real-time PCR results suggest that the relative expression of mRNA level of Galectin-9in transfected group, blank plasmid group and saline controlled group were1.14±0.05,0.46±0.03,0.49±0.07respectively, the mRNA level of Galectin-9is much higher than that in the other two groups(P<0.05), while the mRNA level of T-bet and RORyt is significantly lower in transfected groups compared with the other two groups (P<0.05).(6) The protein level of IFN-γ and IL-17in transfected group, blank plasmid group and saline controlled group were0.39±0.06、1.28±0.11、1.47±0.05and0.64±0.07、1.52±0.05、1.67±0.04respectively, the protein level of IFN-y and IL-17in transfected group is much lower than blank plasmid group and saline controlled group (P<0.05).(7) The serum level of IFN-y and IL-17in transfected group, blank plasmid group and saline controlled group were24.59±9.72pg/ml、121.32±12.77pg/ml、134.54±16.35pg/ml'18.25±7.16pg/ml、87.53±9.28pg/ml、91.62±7.36pg/ml. The serum level of IFN-y and IL-17in transfected group were significantly lower than the other two groups (P<0.05).Conclusion overexpression of Galectin-9can improve the pathological changes of the liver allograft and induce the immune tolerance after rats liver transplantation by eliminated the Thl/Th17cells and related cytokines. |
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