Construction Of The Inflammatory Factor IL-6 High Affinity Receptor Soluble Gp130 In Vitro

Objective:In this work we have constructed recombinant extracellular domain of soluble gp130(sgp130)on the plasmid pcNDA3.1 and expressed it in eukaryotic cells L929.The effect of the recombinant sgp130 protein on the cell proliferation of HepG2,the prevention and treatment of hepatocellular carcinoma in mice,was investaged.Methods:Genetic engineering technology was applied to construct recombinant eukaryocyte expressing plasmid pcDNA-sgp130,which were then transfected into eukaryotic cells L929 for screening the stably transfected cell lines L929-sgp130 through adding G418 in culture media.Sgp130 protein was expressed in L929 cells and purified through affinity chromatography.The following ways were operated to analyze the biological activities of sgp130:the proliteration activity of HepG2 cell was measured through MTT;the apoptosis of HepG2 cell was detected through observing Annexin V-FITC changes;the index of liver function was dectected for liver function evaluation;liver structure was observed through B-mode ultrasonography;the markers of liver was observed through Immunohistochemistry.Results:1.We have successfully constructed recombinant plasmids pcDNA-sgp130,screened the stably transfection cell lines L929-sgp130,and acguired the sgp130 protein purified through nickel column.2.MTT and Annexin V-FITC results showed that sgp130 significantly inhibit the proliferation of HepG2 cell,and enhaned its apoptosis.3.Sgp130 significantly inhibitted the growth and metastasis of tumor tissue in nude mice.4.The results showed that sgp130 protein could significantly reduce the indicatrix concentration of liver function in cancer model mice,such as:ALT,AST,LDH,and so on.5.Analysis of B-mode ultrasonography,hematoxylin-eosin(HE)staining and immunohistochemistry showed that the livers of mice which were injected sgp130 protein had more uniform texture,smoother and more delicated peplos than those of model mice.Sgp130 significantly reduced the degrees of DEN/CCl4 mediated liver fibrosis.6.According to the immunohistochemical staining of liver tissue,TGF-betal,ki-67,VEGF and PCNA were significantly down-expressed in sgp130 protein injected mice.Conclusion:sgp130 protein suppressed IL-6 signal pathway through competitively binding to the IL-6/IL-6Ra complex with gp130,which restrained the growth and proliferation of HepG2,and induced their apoptosis,inhibitted tumor growth,invasion and metastasis.Sgp130 can effectively protect liver from damage induced by diethyl nitrosamine(DEN)/carbon tetrachloride(CCl4),lighten the degree of liver fibrosis,and prevent the development of hepatocellular carcinoma.