Abstract
The proliferation of primary cultured rat hepatocytes was observed in serum-free modified DMEM supplemented with 10mM nicotinamide and 10ng/ml EGF. These proliferating cells were mainly mononucleate and formed small-cell colonies after 4 days of culture. In the present experiment primary cultured hepatocytes were treated with Activin A, IL-1β IL-6, and TGF-β which have been shown to be inhibitors of the DNA synthesis of rat hepatocytes, to examine whether these four inhibitors could suppress the formation of small-cell colonies. The initial DNA synthesis of more than 50% of the cells was dose-dependently inhibited by all the factors and the strongest inhibition was demonstrated in the cells treated with TGF-β. Although Activin A and IL-6 did not block the colony development when the agents were administered at 96 h, just before the time when the cells started to form colonies, TGF-β and IL-β could inhibit the colony formation completely and partially, respectively. Transient treatment (48-72 h) with TGF-β was enough to suppress colony development, while Activin A and IL-6 did not block the formation of colonies. IL-β partially suppressed this formation. However, continuous administration (48-144 h) of IL-β as well as TGF-β stimulated the detachment of the cells from dishes and the remaining hepatocytes failed to form colonies. In addition, only TGF-β could inhibit the DNA synthesis of most small cells in the established colonies as well as that of relatively large hepatocytes. Neither Activin A, IL-β nor IL-6 could inhibit the DNA synthesis of the small cells. Thus, only TGF-β could completely inhibit both the DNA synthesis of any type of hepatocyte and the formation of small-cell colonies.
