Empirical Study Of Therapeutic Effect Of Fetal Liver Stem Cells Modified With Gene On Hepatic Fibrosis

Background: Liver Fibrosis is the result of reversible intercellular extracellular matrix (EXM) deposition resulting from the disbalance of EXM secretion and degradation. It is an inevitable course for many liver diseases progressing to cirrhosis.It is an important turning point for many kind of liver diseases. The prospect of hepatic fibrosis gene therapy is exciting. And the treatment strategy based on cells has been successfully used for end-stage liver diseases,and genetic metabolic liver diseases.Objective: The target gene of our stuty is HGF gene.We will consruct a plasmid that could express HGF, and then transfect this plasmid into hepatic progenitor cell,we will use the hepatic progenitor cells transfected with the plasmid coexpressing HGF and GFP that can be seen with fluorescence microscope to treat hepatic fibrosis cased by CCL4. In our study,we also need to find a good method for purification of hepatic progenitor cell from fetal liver of rats.Methods: We choose the medium-term pregnancy fetal liver of rat in which hepatic progenitor cells are rich and use the immunoabsorption as a major method of separation and purification, and culture hepatic progenitor cells in vitro, analyze the impacts of of cytokines on proliferation and differentiation of hepatic progenitor cells, and then electe the factor(s) which can stimulate proliferation of hepatic progenitor cells as well as inhibit their differentiation.Six group were founded according to the cytokine added into the medium. GroupⅠ: DMEM+10%FCS, GroupⅡ: DMEM+10% FCS+LIF, GroupⅢ: DMEM+10%FCS+bFGF, GroupⅣ: DMEM+10%FCS+LIF+bFGF, GroupⅤ: DMEM+10%FCS+LIF+bFGF+EGF, GroupⅥ:DMEM+10% FCS +LIF+bFGF+SCF; when cells were cultured invitro with the medium and the cytokines, 3H had been added in the medium already, 3H could be absorbed by the cells for DNA synthesis.that is to say, 3H would be found in the cells in the medium after they cultued for coulp of hours.and the ablility of the cells to synthesize DNA was more powerful ,the more 3H would be found in the cells. By this way, we can test the effection of the cytokines on prompe the cells to proliferate. In our study,we constructed a plasmid which can coexpress HGF and EGFP based on plasmid pEGFP-C1.we used liposome to transfect the plasmid vector coexpressing HGF gene and the gene of green fluorescent protein(GFP) into hepatic progenitor cells and confirm the duty of the vector by testing GFP and HGF expression; transplant those cells expressing HGF into the rat of hepatic fibrosis by CCl4, and test the serum indexes of liver function, the concentration of HGF serum TGFβ1 , the volume and distribution of fluorescent protein and typeⅠandⅢcollagen deposition within the liver tissue ,HGF and its receptor and TGFβ1 expression levels in the liver tissue at different time.Results: Getting precursor cells of hepatic parenchymal cells from fetal liver of rats using improved traditional method was proved to be an good strategy for large scale yield of precursor cells of hepatic parenchymal cells.In our study,we gained a satisfactory purification rate,21%. liposome is effective on transfecting pasmid into hepatic progenitor cells .In this way,the hepatic progenitor cells could keep vigorous activity.In the treatment group , liver function of rat were improved significantly (p <0.05). The index of liver function are all better than that of the modle group. green fluorescence was observed in the liver tissue in the treatment group, in the area where the green fluorenscence were conferted,the fibrosis was attenuated and the necrotic cells almost disappeard;the cells in these areas arranged tidily.In liver tissue, cells colony expressing green fluorescent protein formed, in which some cells have the morphologic characteristics of liver cells and biliary epithelial cells, iⅠandⅢcollagen protein in liver decreased significantly in I groups, the intrahepatic level of HGF and it's receptors expression was significantly increased, the expression of TGFβ1 significantly decreased. In serum the expression of HGF slightly increased and that of TGFβ1 declined statistically.Discussion: We had found a new method to treat hepatic fibrosis. Hepatic progenitor cells that having therapeutic effect for hepatic fibrosis were purified from fetal liver of rats by improved traditional method.we constructed a plasmid coexpressing EGFP and HGF, and used liposome to transfect the plasmid into hepatic progenitor cell .The method of transfection with liposome is adapted for plasmid transfection into hepatic progenitor cells.The transfection rate of liposome had been enhanced,and it's traditional predominance were safety and facility.This plasmid expressed the two genes in hepatic progenitor cell,and we found the cells in the livers of receptors by following the GFP expression with confocal microscopy ."in vitro gene therapy" of liver fibrosis is the objective of our study in which we will make some technical optimization on cells preparation. HGF is a kind of anti-hepatic fibrosis cytokines, and its high expression was negatively correlated with liver fibrosis. HGF might raise the expression of its receptors and inhibit TGFβ1which results the EXM deposition. Hepatic progenitor cells from fetal liver cells have a strong targeting effect on liver,they could homing to the liver of receptors after transplantation and then begin to proliferate and differente . The hepatic progenitor cells transfected with the vector containing HGF gene have therapeutic effects on hepatic fibrosis because they can differentiate into normal hepatic cells and generate HGF. The two effects can be promoted mutually to make their co-contribution to reversal of liver fibrosis much more stronger.The effect of LIF+bFGF on Hepatic progenitor cells has two sides,on the one hand, the combinations of the two cytokines is powerful to enhanse the ability of the hepatic progenitor cells to proliferat,on the other hand,they could supress differetiation of the hepatic progenitor cells in the meanwhil.Hepatic progenitor cells which have vigorous proliferation ability are suitable for transplantation, hepatic progenitor cells transfected with pEGFP-C1/HGF, can be in vitro and in vivo express HGF. As soon as the cells were transplanted into rats with hepatic fibrosis , they can treat hepatic fibrosis in two ways at the same time. One is HGF, a kind of anti-hepatic fibrosis cytokine,and the other is hepatic progenitor cells, a kind of cell can differentiate into hepatocyt and bile cut cell.