Studies on the induction and differentiation of rat's hepatic stem cells in vitro / 解放军医学杂志

ABSTRACT

Objective To isolate hepatic stem cells from adult rats,and to examine the process and morphologic changes of isolated hepatic stem cells differentiated into hepatic cells and bile duct epithelial cells under in vitro culturing conditions.Methods AAF/PH was used to stimulate the oval cells.Selective enzymatic digestion and density gradient centrifugation were employed to isolate and purify these cells from heterogeneous liver cell population.The hepatic stem cells were induced to proliferate and differentiate into hepatic parenchymal cells by the addition of dexamethasone,DMSO and several growth factors.The morphologic changes of hepatic oval cells to that of hepatic cells and bile duct epithelial cells were examined.Immunofluorescence method was used to detect the hepatic stem cells c-kit and the expression of OV6 antigen.The albumin of hepatic stem cells and the CK19 expression were identified by RT-PCR analysis.Results The activity of freshly isolated oval cells were over 90%,with different colors and size.The diameter of oval cells was about 1/4~1/6 that of hepatocytes.Large and round hepatocytes appeared first during differentiation,and the cell morphology changed when the oval cells differentiated to hepatocytes.Freshly isolated oval cells was positive for c-kit and OV6 with immunofluorescence staining,and RT-PCR analysis indicated that the albumin and CK19 mRNA were expressed in the new isolated cells.Conclusion The oval cells could be induced to differentiate into a variety of cell lineages including hepatocytes and bile duct epithelial cells in the presence of DMSO and several growth factors.Several antigens such as c-kit,OV6,CK19 and albumin may be expressed in the freshly isolated hepatic stem cells,and the course of morphologic changes may be observed under in vitro conditions of induction and differentiation.