The Therapeutic Effect Of Sgp130 In The Treatment Of Liver Cirrhosis

Objective:In this work we have purified recombinant prokaryotic expression sgp130 protein which were used to immunize Balb/c mice for preparing to sgp 130 monoclonal antibody.We have used the antibody to detect sgp130 protein.Observe the therapeutic effects of sgp130 in cirrhosis C57BL/6 mice induced with ethanol and carbon tetrachloride and DEN-induced cirrhosis ICR mice.Methods:The sgp 130 protein was activated to induce expression in constructed prokaryotic expression strain pET-28a-sgp130-Rosseta(DE3)and purified.The EDTA antigen repair solution heat pprepaired protein was used as an antigen to immunize Balb/c mice to prepare polyclonal antibodies.Hybridoma cells were prepared by cell fusion technique and the antibody was screened and evaluated using ELISA and western-blotting methods.The antibody isoforms were determined by ELISA using a subtype identification kit.A liver cirrhosis model of C57BL/6 mice was established by the combination of alcohol and carbon tetrachloride,and a cirrhosis model was induced by DEN I in ICR mice.The purified sgp130 protein was injected into the cirrhosis model mice via the tail vein,respectively.After the treatment,the mice hepatic pathological changes were detected by Sirius red staining,HE staining,and immunohistochemistry.Results:1.The sgp130 protein was successfully expressed and purified.The immune titer in immunized Balb/c mice reached 1.25×105 after being immunized three times.2.The sgp130 hybridoma cells were successfully prepared,and 4 lines of hybridoma cells producing high level antibodies were obtained which western-blotting results showed good specificity and no background staining of the antibodies.3.The subtype identification results showed that the antibody heavy chain subtypes are IgG1,and the light chain subtypes are κ type.4.The results of dissection,hepatic HE staining and Sirius red staining showed that the hepatic texture was uniform,the capsule was smooth and slender,liver fibrosis was significantly reduced,and liver damage was significantly reduced in sgp130 treated cirrhosis mice.5.Immunohistochemical detection of liver tissue showed that the expression of Ki67 and PCNA in the liver of mice injected with sgp130 protein was significantly reduced.Conclusion:The sgp130 monoclonal antibody was obtained with high specificity and high affinity which screen out with the western-blotting assay.The sgp130 protein inhibited the trans signaling pathway of IL-6 activation and reducing the degree of inflammatory reaction through competing with the gp130 protein to bind to the IL-6/IL6Rα complex,.Intravenous injection of sgpl30 not only reduced hepatic fibrosis induced by ethanol and carbon tetrachloride but also can reduce the damage caused by DEN and CCl4.