| Objective:To construct the adenoviral vector that expresses fusion gene encoding Hyper-IL-6,and the controls that express IL-6 or adenovirus not containing objective gene.To package and propogate the three recombinant adenoviruses respectively ,then to explore the treatment effects and mechanisms of Hyper-IL-6 mediated by recombinant adenovirus on rat with acute liver failure(ALF).Methods:The adenoviral shuttle plasmid containing Hyper-IL-6 was digested with Pme-I.The linearized shuttle plasmids were co-transformed with adenoviral backbone vector to E.coli BJ5183 cells. The candidate clones were further tested by restriction nuclease digestion with Pacâ… and PCR. Before transfection, the recombinant adenoviral plasmids were digested with PacI. Then the digested recombinant adenoviral plasmids were transfected to 293 cells with liposome for package and amplification of adenoviruses,then the viruses were purified through CsCl density-gradient centrifugalization .The control viruses were generated with the same method. The ALF rat model was induced by D-Gal ,and eighty SD rats were randomly divided into 4 groups(each 20) after 6h injected D-Gal:normal saline was injected for non-infection group ;Ad0,AdIL-6 and AdHIL-6 was respectively injected for group Ad0,group AdIL-6,group AdHIL-6.Sequently each group was again divided into group A(n=10) and group B(n=10).Group A was observed for 3 days,then the blood samples were collected to determine the index of hepatic function(ALT,TB),the liver tissues were taken for H-E staining,immunohistochemistry of PCNA and Caspase-3,and to detect the hepatocelluar apoptosis in situ by TUNEL assay.Group B was kept observing for 14 days in order to conclude the survival rate of rats.Results:The object staps about 3kb and 30kb emerged after the recombinant adenoviral plasmids were digested with Pac-I,which demonstrated successfully homologous recombination.After transfection, the brighter green fluorescence was observed in 293 cells.The viruse liquid can be amplified object genes:IL-6,Hyper-IL-6,AdEasy-1.The contents of serum ALT and TBil,and apoptotic index in group AdHIL-6 were markedly decreased than that of the other groups(P<0.01),inversely the significant expression of PCNA(P<0.01) .The state of liver necrosis was terrible,and extremely positive expression of Caspase-3 was in non-infection group and group Ad0.The extent to liver injury was slightly reduced than that of the former 2 groups,and highly positive expression of Caspase-3 in group AdIL-6.The liver pathological change was the most mild,and positive expression of Caspase-3 in group AdHIL-6.At the end of 14 days after injecting D-Gal,all rats died in non-infection group and group Ad0,there was only one alive in group AdIL-6 ,however 3 rats stayed lively in group AdHIL-6.Conclusion:The generating of the three recombinant adenoviruses was successful. It was significant that the potency of Hyper-IL-6 enhancing hepatocelluar proliferation and defending hepatocyte apoptosis on the rats with ALF. Hyper-IL-6 can effectively ameliorate liver function and histology,and improve the survival of rats with ALF. |
