|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1995 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1994 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1993 : ¥700,000 (Direct Cost : ¥700,000)
1. Hepatocytes were isolated from the livers of adult rats. After plating to plastic dishes, the hepatocytes were incubated in serum-free Williams' medium E (WME) overnight at 37ﾟC.After incubation, the hepatocytes were cultured in serum-free WME supplemented with epidermal growth factor (EGF) and insulin (Medium 1), EGF,insulin and dexamethasone (DEX) (Medium 2) or EGF,insulin and dimethylsulfoxide (DMSO) (Medium 3) for 2 hr. After termination of the culture, the hepatocytes were fixed and stained to alphafetoprotein (AFP), glutachione S-transferase P (GST-P) or c-myc by use of a immunocytochemical technique. The hepatocytes cultured in Medium 1 or Medium 3 showed feeble positive reactions to c-myc, whereas the hepatocytes cultured in Medium 2 show ed no positive reaction to it. The hepatocytes cultured in any medium did not show positive reactions to GST-P or AFT.The results in this study seem to indicate, in cultured adult rat hepatocytes, that though the expression of c-myc, GST-P
or AFP is a feeble level, c-myc reacts more sensitively to EGF than GST-P or AFP and is expressed more early than other carcino-embryonic proteins.
2. Hepatocytes were isolated from the livers of fetal rats at day 17 of gestations. The cells were incubated in WME supplemented with 20% fetal calf serum overnight. After incubation, themedium was replaced with the following four kinds of media : Medium 1 consisted of WME supplemented with EGF and 20% fetal calf serum. Another medium consisted of Medium 1 supplemented with DEX (Medium 2), DMSO (Medium 3) or sodium butyrate (Medium 4). Subsequently, the cells were cultured for 3 or 6 days. After termination of the culture, the monolayrs of the attached hepatocytes were fixed and stained to AFP,GST-P or c-myc by use of a immunocytochemical technique. Hepatocytes cultured in Medium 1 showed strong positive reactions to AFP but negative reactions to GST-P and c-myc. On the other hand, positive reactions to AFP were not observed in hepatocytes cultured in Medium 2 or Medium 4, but hepatocytes cultured in Medium 3 showed positive reactions to it. The results in this study indicate, in cultured fetal hepatocytes, that AFP is expressed in a stronger level than other carcino-embryonic proteins and that DEX and sodium butyrate supress the expression of alpha-fetoprotein but DMSO does not suppress it. Less