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

To construct pEGFP-hepatic growth factor (HGF) expression vector, detect its transient expression in transfected human hepatocytes, and to investigate the influence of autocrine HGF expression on the proliferative potential and cytoprotective effects in human hepatocyte.

Human HGF cDNA was ligated to pEGFP vector. The recombinant plasmid was transfected into human hepatocyte line QZG with liposome. The expression of HGF protein was observed by fluorescence microscopy and immunohistochemistry. Hepatic cells were collected 24, 48, and 72 hours after transfection to detect the number of [(3)H]-TdR uptake in DNA. DNA synthesis was observed by using PCNA stain immunohistochemistry. Acute liver cell damage was induced by carbon tetrachloride. The supernatant of culture 10 days after transfection of pEGFP-HGF was collected to put in the normal culture of hepatic cells. Another sample of supernatant was added with anti-HGF antibody to block the HGF activity as control. Cytoprotective effect was observed by examining the survival rate of hepatocytes and leakage of intracellular alanine transaminase (ALT) and potassium ions.

HGF identification of pEGFP-HGF by enzyme digestion showed that HGF fragment had been cloned into BamH I and Sal I sites of pEGFP-N3. Expression of GFP in transfected hepatocytes was observed with fluorescence microscopy. The [(3)H]-TdR uptake became 7 times as much as in the control group 96 hours after transfection. After HGF transfection, survival rate of hepatocytes poisoned by CCl4 significantly increased (83% vs 61%, P < 0.05), and leakage of intracellular alanine transaminase and potassium ions decreased (35.16 U x L-1 vs 65.31 U x L-1, P < 0.01; and 5.59 mmol/L vs 6.02 mmol/L, P < 0.01 respectively). Culture of transfected hepatic cells promoted the proliferation of other non-transfected cells. This effect was blocked by anti-HGF antibody.

Transfected HGF is expressed in hepatic cells and has the activity of promoting cell division and protecting hepatic cells against poisoning.