Adenoviral Delivery Of Truncated MMP-8 Fused With The Hepatocyte Growth Factor Mutant 1K1 Ameliorates Liver Cirrhosis And Promotes Hepatocyte Proliferation

Background:Liver cirrhosis is a typical feature of most chronic liver disease. It is a protective effect against chronic liver damage, and it is a reversible scar formation reaction. In many patients, the cause of liver cirrhosis is unclear. The probable inducement including: virus, autoimmunity, drug, alcohol, cholestasis and metabolism. Its clinical manifestation is changeable, from no manifestation detecting by experimental examination to liver function failure of later stage. Liver cirrhosis accounts for the high levels of morbidity and mortality associated with this disease worldwide.In the current literature, the primary focus concerning cirrhosis is on prevention and the basic underlying mechanisms of the disease. Although some studies have investigated treatments to promote hepatocyte proliferation, inhibit HSC activation and/or function, and degrade ECM during liver-related wound-healing responses. Another part, liver transplantation and control the primary disease is also very important.Objectives:In our study, we constructed a fusion protein cMMP8-1K1 with protein engineering, increasing infection and distribution in liver with adenoviral vector. We injected adenovirus into 70% hepatectomy and liver cirrhosis mouse models to find the function of fusion protein on liver cirrhosis and discussed the mechanism of fusion protein on liver cirrhosis in vitro according the bio-information provided by in vivo experiment. We look forward to find a new strategy for liver cirrhosis.Methods:1, We constructed a fusion gene with chemically synthesize, and obtained adenovirus with adenoviral vector containing fusion gene.2, We injected fusion protein adenovirus/negative control adenovirus into 70% hepatectomy and liver cirrhosis mouse models to find the function of fusion protein on 70% hepatectomy and liver cirrhosis by detecting the distribution of adenovirus in mouse, dyeing, immunoblotting, ELISA methods on liver, serum and other tissues.3, Based on in vivo experiment, we discuss the mechanism of fusion protein on liver cirrhosis in vitro by detecting cell proliferation, apoptosis, immunoblotting, PAGE on cells and ECM, such as HL-7702 cells, LX2 cells, type I collagen.Results:1, We constructed a fusion gene with chemically synthesize, and obtained adenovirus with adenoviral vector containing fusion gene. The gene sequence is correct, and the virus titer confirms the need of experiment.2, We injected fusion protein adenovirus/negative control adenovirus into 70% hepatectomy and find the fusion protein promotes the proliferation of hepatocytes of 70% hepatectomy and protects liver function.3, We injected fusion protein adenovirus/negative control adenovirus into liver cirrhosis mouse models and find the function of fusion protein can decrease the expression of α-SMA, TGF-β 1 and collagen deposition which provided the amelioration of liver cirrhosis morphologically and functionally, promote expression of PCNA, protect liver function.4, In vitro, fusion protein can promote the proliferation of HL-7702 cells by phosphorylation of c-Met, Mek, erk to increase the expression of PCNA. Furthermore, it can inhibit the mRNA of TIMP-1、α-SMA、TGF-β1. COL I/III, and induce the apoptosis in LX2 cells.5, Additionally, fusion protein also can degrade soluble type I collagen (MW=120KD) into two separate part:85KD and 35KD in vitro.Conclusion:1, Fusion protein injected by adenovirus vectors can concentrate in liver, and promote liver proliferation, amolerate liver cirrhosis and protect liver function.2, In vitro, fusion protein can promote hepatocyte proliferation by phosphorylation of c-Met, Mek, erk. Furthermore, it can inhibit the mRNA of TIMP-1、α-SMA、TGF-β1、COL I/III, and induce the apoptosis in LX2 cells.3, Additionally, fusion protein also can degrade soluble type I collagen provide a direct collagen degradation function.