Development and characterization of an in vitro model for liver homeostasis.

Abstract

The major objective of this work was the development of an in vitro model for liver homeostasis which would allow the future study of early events in cell proliferation and cell death. The model which was set up involves growing T51B rat liver epithelial cells with a single dose of 1nM epidermal growth factor (EGF). This results in a period of hyperplasia where the cells reach double the control cell numbers two days after EGF addition. This is then followed by a decrease in cell numbers and the cell density returns to around the confluent control level five days after EGF addition. The model was investigated to ascertain whether the decrease in cell numbers three to five days after EGF addition was due to an increase in apoptosis. The results from light and electron microscopy studies, from the electrophoresis of T51B cell DNA and from the quantification of nuclear fragmentation indicated that the cells do die via an increase in apoptosis. The electron microscopy studies also show that healthy T51B cells can phagocytose apoptotic bodies. This suggests that the model is more physiological than other in vitro models of apoptosis. Cell growth studies and EGF binding studies were carried out in order to try to determine which events, if any, are EGF specific. The results from these studies suggest that occupancy of the low affinity binding site of the EGF receptor is responsible for the hyperproliferation seen when the T51B cells are grown with high doses of EGF. These studies also suggest that the apoptosis could be triggered by the down-regulation of the receptor, in a manner analogous to the removal of a trophic hormone in other systems. Thus this work describes the development and characterization of an in vitro model of liver homeostasis which closely parallels in vivo systems where animals are given mitogenic stimuli, and it also provides a good system for studying the role of EGF in cell proliferation and apoptosis.