| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1992: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1991: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
We found previously that mouse hepatocyte growth factor (mHGF) in serum and liver markedly increased prior to liver regeneration in carbon tetrachloride-treated mice. mHGF was then purified about 280,000-fold from the liver supernatant of mice treated with the hepatotoxin and was characterized. It is composed of two peptide chains with molecular masses of 62 and 31 kDa, linked by a disulfide bond(s). Its physicochemical and biological properties are quite similar to those of hHGF. In this study, we found that homogenizing the damaged liver with 1.7 M NaCl (pH 7.4) in phosphate-buffered saline (PBS) extracted 5-10-fold more mHGF than homogenization of the liver with 0.25 M sucrose containing 20 mM Hepes (pH 7.4). mHGF, extracted by this modified method, was purified by our four-step purification method. We next examined whether D-galactosamine-induced damage of rat hepatocytes in primary culture is prevented by adding mHGF or hHGF to the hepatocyte cultures. mHGF or hHGF did not diminish the hepatotoxicity of D-galactosamine at doses of 10-30 ng/ml, which induce DNA synthesis in hepatocytes. Effect of hHGF on the growth of various cultured cells was next investigated. It stimulated proliferation of HuH-6 Clone 5 human hepatoblasstoma cells, but did not that of HLE, HuH-7, and PLC/PRF/5 human hepatoma cell lines. The growth of Hep G2, another human hepatoblastoma cell line, was inhibited by hHGF. Studies on other hepatoma and hepatoblastoma cell lines are required to determine whether hepatoblastoma, and not hepatoma cells are responsive to hHGF. It has not been clear whether hHGF affect the growth of fibroblasts, but in this study we demonstrated significant mitogenic activity of hHGF for human skin fibroblasts. We reported previously that the fibroblasts secreted a small but detectable amount of hHGF. Furhter studies are required to answer the question of whether proliferation of human skin fibroblasts is stimulated by hHGF in an autocrine manner.
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