The effect of cell morphology on differentiation and proliferation of adult rat hepatocytes

摘要

We studied the spheroid formation (multi-cellular aggregates formation) of hepatocytes and the expression of liver specific functions such as albumin secretion when hepatocytes were cultured with various extracellular materials. Poly-D-lysine and Eudragit induced spheroid formation and hepatocytes in spheroids exhibited higher liver specific functions compared to monolayer culture. The results indicated that cell-cell interaction caused by spheroid formation was a key factor to promote the expression of liver specific function. We then examined the relationship between several transcription factors and liver specific function during the spheroid formation. Among transcription factors, the expression of CCAAT enhancer binding protein (C/EBPalpha) increased and the high expression level was maintained during the culture when hepatocytes formed spheroids. On the other hand, HGF response ability was repressed when hepatocytes formed spheroids. The signal caused by HGF stimulation was transmitted to its receptor c-Met in the cells of both monolayer and spheroid; the phosphorylation of c-Met occurred. However, the ratio of GTP-bound Ras after HGF addition was different between monolayer and spheroid; Ras signal pathway was blocked in spheroid. We also examined the role of Cdk inhibitors (p21 and p27) in hepatocyte culture and the results revealed that the Cdk inhibitors were expressed high level in spheroid and that hepatocytes in spheroid maintained G_0 phase and entered highly differentiated state by the activity of the Cdk inhibitors.