|Budget Amount *help
¥6,700,000 (Direct Cost : ¥6,700,000)
Fiscal Year 1994 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1993 : ¥4,700,000 (Direct Cost : ¥4,700,000)
MRC-5 cells were plated on 24-well plastic dish in 0.5 ml of DMEM supplemented with 10% FCS and antibiotics, and cultured at 37 ﾟC in a humidified atmosphere of 5% CO_2 in the presence or absence of patient's serum. Serum samples were obtained from normal subjects, patients with fulminant hepatic failure (FH), acute hepatitis (AH), chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Serum samples were added to the culture medium at a final concentration of 5% and the cells were cultured for 24 hours after the addition of samples. Hepatocyte growth factor (HGF) in the culture supernatant was determined using an enzyme-linked immunosorbent assay (ELISA) and HGF production was expressed as HGF amount per cellular protein (ng/mg protein).
Sera from patients with AH and CH increased HGF production in MRC-5 cells with dose- and time-dependent manner. The relationship between HGF production by patients' serum and HGF levels in serum of AH and CH patients was positively significant. Northern blot analysis revealed an increase in HGF mRNA levels of MRC-5 cells cultured with serum from AH patients. HGF-inducing activity in the patients' serum was disappeared by heat treatment (56ﾟC,30min) and treatment with trypsin or chymotrypsin. We also examined the effects of interleukin-1 (IL-1), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), phorbol ester (PMA), dexamethasone and heparin on HGF production in MRC-5 cells cultured with serum having a high HGF- inducing activity. IL-1, TNF-alpha and PMA had an additive effect on HGF production stimulated by patient's serum in MRC-5 cells, however, dexamethasone suppressed the HGF production.
In summarize, HGF-inducing activity was present in serum of patients with various liver diseases and this activity may be a physical HGF inducer in humans.