Autocrine expression of hepatocyte growth factor and its cytoprotective effect on hepatocyte poisoning

AIM:To construct pEGFP-hepatocyte growth factor(HGF)expression vector,the to detect its expression in transfectedhuman hepatocytes,and to investigate the influence ofautocrine HGF expression on the proliferative potential andcytoprotective effects in human hepatocytes.METHODS:Human HGF cDNA was ligated to the pEGFP vector.Recombinant plasmid was transfected into human hepatocyteline QZG wibh liposome.Expression of HGF protein was observedby fluorescence microscopy and immunohistochemistry.Hepaticcells were collected 24,48,and 72 h after transfection todetect the number of[~3H]-TdR uptake in DNA.DNA synthesiswas observed by using PCNA stain immunohistochemistry.Acute liver cell damage was induced by carbontetrachloride.Cytoprotective effect was observed byexamining the survival rate of hepatocytes and leakage ofintracellular alanine transaminase(ALT)and potassium ions.RESULTS:HGF identification of pEGFP-HGF by enzymedigestion showed that HGF fragment was cloned into BamHI and Sa/I sites of pEGFP-N3.Expression of GFP in transfectedhepatocytes was observed with fluorescence microscopy.The[~3H]-TdR uptake became 7 times as many as in thecontrol group 96 h after transfection.After HGF transfection,the survival rate of hepatocytes poisoned by CCI4significantlyincreased(83% vs 61%,P<0.05),and the leakage ofintracellular alanine transaminase and potassium ions decreased(586 nkat/L vs 1089 nkat/L,P<0.01;and 5.59 mmol/L vs6.02 mmol/L,P<0.01 respectively).Culture of transfectedhepatic cells promoted the proliferation of other non-transfected cells.CONCLUSION:Transfected HGF is expressed in hepaticceils and has the activity of promoting cell division andprotecting hepatic cells against poisoning.