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  • Title: The stimulation by epidermal growth factor (urogastrone) of the growth of neonatal rat hepatocytes in primary tissue culture and its modulation by serum and associated pancreatic hormones.
    Author: Draghi E, Armato U, Andreis PG, Mengato L.
    Journal: J Cell Physiol; 1980 Apr; 103(1):129-47. PubMed ID: 7000798.
    Abstract:
    Epidermal growth factor (EGF) added in a single dose (between 10(-16) and 1.7 X 10(-9)M) to neonatal rat hepatocytes in primary culture with subsequent incubation for 12 and 24 hours in Eagle's MEM fortified with 10% (v/v) FBS stimulated their entry into S and M phases, as shown by (3H)thymidine labeling and autoradiography and by a 4-hour exposure to colchicine (0.1 mM). Growth stimulation by EGF was detectable after 4 hours, peaking between 12 and 16 hours, and thereafter declining in intensity. Rat hepatocytes exposed for 72 hours (between the fourth and the seventh day in vitro) to no serum or to 10% fresh FBS possessed similar growth rates and absolute numbers in the cultures. A 24-hour exposure to 20 to 50% FBS stimulated hepatocytic DNA synthesis and mitotic activity and resulted (except for the 50% FBS treatment) in increased hepatocytes' numbers, which were relatively greater than the concurrent increases in connective tissue cell numbers. In serum-devoid medium EGF (10(-11)M) enhanced hepatocytic mitotic, but not DNA-synthetic activity. To be fully effective EGF required a 10% FBS addition to the medium, then eliciting within 24 hours a marked increase in hepatocytes' number with respect to cultures incubated with 10% serum only. When associated with 20 to 30% FBS, EGF stimulated parenchymal cell growth at rates slightly higher, but not significantly different, than those elicited by the same serum concentrations alone. However, when used in conjunction with 10 to 30% FBS, EGF preferentially increased the number of hepatocytes rather than that of non-parenchymal cells. Moreover, comparative proliferation kinetic studies showed that in the presence of 10% FBS, an equimolar (10(-14)M) mixture of EGF, insulin, and glucagon promoted an early and marked increase in the DNA-synthetic and mitotic activities of hepatocytes, which peaked after 8 hours. Within a 24-hour time lag this growth stimulation was as effective in increasing the final hepatocytes' number as was a 1000-fold higher EGF concentration, and was twice as active as either an equimolar (10(-14)M) mixture of the two pancreatic hormones or EGF by itself at 10(-14)M. These results show that the growth-promoting effect of EGF on primary neonatal rat hepatocytes is modulated by serum factor(s) and can be additively amplified by the simultaneous administration of subphysiological doses of glucagon and insulin.
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