Isolation And Purification Of A Novel Human Hepatic Growth Factor: Hepatopoietin Cn And Study On Its Biologic Activity

In our country, it's general that people have liver diseases. As the quickly development of technology and economy, people's living rhythm has speeded up, also, their health has aggravated. The incidence of hepatitis, hepatic cirrhosis and liver cancer has been higher and higher, and it has seriously harmed our society. So it is great significance to study hard on the mechanism of molecular biology of hepatic injury and to develop new drugs of curing liver diseases. At this background, a novel human hepatic growthfactor—HPPCn was generated. It's a monomer which can specificallypromote the proliferation of SMMC 7721.In this study, our main purpose is to purify HPPCn- recombinant expression product and to have a founction study on the purified product in vivo and vitro. HPPCn was purified by two steps anion-exchange chromatography according to its characteristics like isoelectric point. The concentration of purified HPPCn was detected by method Bradford, and its purity coefficient was analysised by HPLC and gel-scanning. Its immunogenicity was analysised by Western blot and it was identified by MALDI-TOF. As a result, the purity coefficient of HPPCn after two steps anion-exchange chromatography reached 94% , and the total yield of purification was 8.5%. The total protein and its purity coefficient all reached the requirement of activity detection in vivo and vitro.Promoting proliferation activity of purified HPPCn to SMMC7721 was detected by method MTT and ³H-TdR DNA incorporation. The rat original hepatocyte was isolated by rat liver-perfusion in situ, to which proliferation activity of purified HPPCn was detected by ³H-TdR DNA incorporation. The result indicated that purified HPPCn can significantly promote the proliferation of SMMC7721 and rat original hepatocyte.Finally, the activity in vivo of HPPCn was studied. HPPCn was verified to have close relation-ship with liver regeneration by detecting variation of HPPCn expression in injuried liver in classic partially hepatectomized( PH); CCl₄ and ConA inducing mouse acute injury of liver and CCl₄ combination inducing rat hepatic fibrosis were constructed, and the variation of serum biochemical indicator and liver pathological section were detected. The result indicated that HPPCn could significantly cure and repair the acute or chronical hepatic injury. In all these animal models, there was a phenomenon that groups given HPPCn bled obviously than groups given saline. The model of chick embryo allantocherion was constructed to detect whether HPPCn could promote vascularization. and the result indicated that HPPCn could not promote vascularization.