Construction Of HGF Expression Vector And Its Treatment For Hepatic Fibrosis

Hepatocyte growth factor (HGF) is a kind of growth factor with many functions.As a exogenous growth factor, it can promote cell proliferation, cell movement, anti-apoptosis and anti-cell differentiation It participates proliferation, movement and morphopoiesis of many kinds of cells and has effect of nutrition recovery on many kinds of organ and tissue and improve impaired organ regeneration, such as liver,kidney, lung,myocard, skeleton, hematopoiesis. Meanwhile it is one of new nutrition factors of nervous system. As a strong mitogen and liverphilc factor of mature hepatocellular, HGF can improve parenchyma hepatocellular regeneration remarkably when partially hepatectomized and hepatonecrosis .It has been reported that HGF can slow down the development of acute fulminant hepatitis, prevent chronic hepatitis from developing to cirrhosis and improve the clinical symptom of cirrosis and HGF had effect on chronic liver disease and progressing cirrhosis. It became the hot point of liver disease research. During the course of hepatic fibrosis formation, the relative capacity and absolute number of hepatocellular decreased. So to stimulate and improve liver regeneration is also important strategy and measure of prevention of hepatic fibrosis.Recent yeares the development mechanism of apoptosis was researched deeply. The close relationship between many liver disease and caspase family was recognized. Cysteinyl aspartate specific protease (caspase) that is special to aspartate has key effect during the course of apoptosis. Caspase was found increased during all stage of apoptosis.This article is to approach the effect of HGF gene to hepatocellular growth and generation ,anti-impairment capability,to animal model of hepatic fibrosis, and to the mechanism of liver HSC and hepatocellular apoptosis. This article is to provide subject and theory foundation for gene therapy of hepatic fibrosis and other liver disease. Our experiment did some research and got propotional experimental results as follows:1. We used gene cloning recombinated technology and constructed and identified eukaryotic expression vector PCI-neo-HGF with human hepatocellular growth factor gene by restriction enzyme digestting and gene sequencing. The homology of HGF cDNA was 99% by contrasted with HGF gene sequence of GenBank. Recombinated eukaryotic expression vector was transfected into COS7 cells and was expressed after 12h. The expression was strongest on the 36-72h (2000ng.L-1) and decreased. The inhibition ratio was still high with descent after 72h.2. Human hepatocellular growth factor eukaryotic expression vector PCI-neo-HGF was transfected into HL-7702 cells with liposome. HGF was proved that HGF had expressed by SP immunity histochemistry and western blot.3. Compare biology activity of HL-7702 cells into which PCI-neo-HGF was transfected and HL-7702 cells, HL-7702 cells which were cultured with the culture media of COS7 cells with HGF and HL-7702 cells by MTT assay. The result showed HGF had great promotion effect for growth and proliferation of normal hepatocellular (HL-7702).4. This experiment also chose HL-7702 cells and HL-7702 cells transfected with PCI-neo-HGF and used CCl4 to induce HL-7702 cells to apoptosis and to stimulate CFSC-2G cells. Caspase-3, caspase-8 and caspase-9 of HL-7702 cells and CFSC-2G cells into which PCI-neo-HGF was transfected were dectected. The results showed caspase-3 expression of HL-7702 cells into which PCI-neo-HGF was transfected was much less than HL-7702 cells which were injured by CCl4 and HL-7702 cells into which PCI-neo was transfected and caspase-8 and caspase-9 were not found. Caspase-3 expression of CFSC-2G cells into which PCI-neo-HGF was transfected was more than CFSC-2G cells which were injured by CCl4 and caspase-9 was found. The results suggested that PCI-neo-HGF transfected into HL-7702 cells could antagonize apoptosis of HL-7702 cells whose mechanism might be related with caspase-3 expression down regulation and could promote rat CFSC-2G cells apoptosis whose mechanism might be related with caspase-3 and caspase-9 expression up-regulation..5. We constructed rat animal model of hepatic fibrosis by CCl4 which was confirmed by hepatic tissue pathology detection and transfected the recombinated eukaryotic expression vector to them in vivo by injection to mescles of right lower limb. The results confirmed that HGF gene that was transfected to the animal model was expressed in vivo by SP immunity chemistry assay and ELISA assay and had some protective effect on hepatocytes and therapeutical effect on anti-hepatic fibrosis by detection of hepatic fibrosis four indexes. The experiment found survival rate of treatment group of transfection with PCI-neo-HGF, preventive medication group and treatment group1 was raised, liver fuction was determinately and the pathohistology was better than model group. Fibroplasias was less, the formation rate of pseudo-lobule was lower and the degree was lighter. We didn't find typical pseudo-lobule. Fatty degeneration of hepatocellular was decreased. The pathological change of part rats were not obvious and similar to normal hepatic tissue. Compared with other groups, fibroplasia of preventive medication group was less, limited in portal area of liver tissue, the main pathological changes was hepatocyte fatty degeneration and pseudo-lobule formation rate was lower. The improvement rate of hepatic tissue was high near to Dansshen group. The results indicated that transfection with PCI-neo-HGF had therapic effect of hepatocyte production and anti-hepatic fibosis and was worth doing more research.