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  • Title: Effects of mitogens and co-mitogens on the formation of small-cell colonies in primary cultures of rat hepatocytes.
    Author: Mitaka T, Norioka K, Nakamura T, Mochizuki Y.
    Journal: J Cell Physiol; 1993 Dec; 157(3):461-8. PubMed ID: 8253857.
    Abstract:
    Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free Dulbecco's modified Eagle's medium containing 10 mM nicotinamide and 10 ng/ml of epidermal growth factor (EGF), acidic and basic fibroblast growth factors (FGF), hepatocyte growth factor (HGF), or transforming growth factor-alpha (TGF-alpha). Every colony consisted of cells that each had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically the cells induced by any mitogen were stained with albumin, transferrin, cytokeratin-8 and -18. But these cells expressed neither cytokeratin-7 nor -19. When 6 x 10(5) cells were plated on 35-mm dishes, about 15 colonies per 1,000 attached cells were observed in the cultures treated with EGF, HGF, and TGF-alpha. Although FGFs could also induce colonies, their number was less than half of the number induced by EGF. Furthermore, the numbers of colonies induced by the combinations of EGF+HGF, EGF+TGF-alpha, and HGF+TGF-alpha were not different from those of the colonies induced by each mitogen alone. To examine the ability of co-mitogenic factors to induce small-cell colonies, angiotensin-II, insulin-like growth factor-I, norepinephrine, tumor necrosis factor, and vasopressin were used. In the cells cultured without EGF, these co-mitogens neither stimulated DNA synthesis nor induced colonies. On the other hand, in cells cultured with both EGF and each co-mitogen, although the DNA synthesis of the hepatocytes was enhanced, the number of colonies detected was not significantly different from the number which EGF alone could induce. These results showed that the small-cell colonies in primary cultures of rat hepatocytes were inducible by EGF, HGF, TGF-alpha, or FGFs and that the co-mitogens did not influence the formation of the small-cell colonies.
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