The Effect On Phenotypes Of Hepatic Cells By Substrate Rigidity And TGF-β1

Objective: To explore the mechanical-chemical synergistic effects of substraterigidity and the cytokine TGF-β1on the phenotypic transformation of hepatocytes,through the establishment of an in vitro cell culture model with an physiologically andpathologically relevant as well as controllable substrate rigidity.Methods: Polyacrylamide substrates with different rigidity (3.6kPa、10kPa、30kPa)were prepared by controlling the concentration, hydrophobicity as well as the degree ofcross-linking of the hydrogel. We determine the optimum concentration ofTGF-β1through the concentration gradient experiment. After that hepatocytes werecultured on the substrates for3days before cell morphology migration and cytoskeletalorganization were evaluated through microscopic observation and image analysis. Theexpression of phenotypic markers E-cadherin, albumin and alpha-Smooth muscle actinwere characterized with Western-Blotting and PT-PCR.Result: The experiment was performed after72hours, The spreading area and thelong/short(L/S) axis ratio was positively correlated with substrate rigidity. Thecytoskeleton which was assembled as cortical ring in cell periphery on soft substratechanged to the state of rich stress fiber and parallel to the long arix. There were moreabundant expression of E-cadherin and albumin, but the less expression of alpha-SMAin soft substrate as compared to high rigidity substrate groups through Western Blottingand RT-PCR. But the expression of alpha smooth muscle actin trends to the contrary. Inthe same rigidity substrate, the phenotypic transformation of hepatocytes furtherincreased with adding TGF-β1, and E-cadherin and albumin expression decreased bothin its protein level or DNA level (compared with controls), but alpha smooth muscleactin expression increased.Conclusion: The increasing of substrate-rigidity can induce hepatocyte phenotypetransformation and promote the impact of TGF-beta1on hepatocyte metabolicbehavior.