Recovery of mRNA expression of tryptophan 2,3-dioxygenase and serine dehydratase in long-term cultures of primary rat hepatocytes.
Expression of tryptophan 2,3-dioxygenase (TO) and serine dehydratase (SDH) has not previously been maintained or re-induced in long-term cultured hepatocytes. In the present study, we succeeded in inducing expression of TO and SDH mRNAs in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor and 2% dimethyl sulfoxide (DMSO). After the start of culture, the expression of TO mRNA rapidly disappeared and at 96 h it was less than 10% of that at isolation. However, after the addition of 2% DMSO from 96 h, the transcript level gradually increased and reached about 40% of that of the isolated cells at day 14. In addition, the expression of TO mRNA was enhanced in cells treated with both 10(-5) M dexamethasone and 10(-7) M glucagon. In contrast, the expression of SDH mRNA decreased very rapidly and we could not detect it after 24 h of culture. Furthermore, 2% DMSO failed to induce it. However, when both 10(-5) M dexamethasone and 10(-7) M glucagon were added to the culture medium at day 9, we observed dramatic induction of SDH mRNA 24 h later. Primary hepatocytes cultured by this method could express and maintain highly differentiated hepatic functions for a long time. Thus, this in vitro system is suitable for the investigation of hepatic functions.
Mizuguchi
T
T
Department of Pathology, Sapporo Medical University School of Medicine.
Mitaka
T
T
Kojima
T
T
Hirata
K
K
Nakamura
T
T
Mochizuki
Y
Y
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005934:Glucagon / Q000494:pharmacology; D007700:Kinetics; D012695:L-Serine Dehydratase / Q000096:biosynthesis*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012709:Serum Albumin / Q000096:biosynthesis; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014366:Tryptophan Oxygenase / Q000096:biosynthesis*
D012333:RNA, Messenger; D012709:Serum Albumin; D003907:Dexamethasone; D005934:Glucagon; D014366:Tryptophan Oxygenase; D012695:L-Serine Dehydratase; D004121:Dimethyl Sulfoxide