Study Of The Induction And Differentiation Of Hepatic Stem Cells Into Insulin-Producing Cells

To explore the induction and differentiation of hepatic stem cells into insulin-secreting cells, we use methods of immunohistochemical staining, cell culture, molecular clone technology, SDS-PAGE, ELISA and RIA to study the distribution and migration of hepatic stem cells, as well as the mechanism of their differentiation into islet P-cells.All the experiments are divided into four parts.Part 1: Histochemical study of human fetal hepatic stem cells. Objective In this study, we introduced an immunohistochemical method to label the unique cells during various stages of development with the purpose of investigating the hepatic stem cells' distribution and morphology of the normal in human fetal livers and exploring occurrence and development of liver and the origin of hepatic stem cells. Methods A biliary epithelial cell expressing protein CK19 and the hematopoietic stem cell marker CD34 were performed ABC staining. Meanwhile, the rat oval cell marker OV6 and a polyclonal antibody C-l 1, which is always expressed on the ductal plate cells, were used to immunostain the progenitor cells in the fetal livers. Results The hepatic stem cells lying in the limiting plate monolayer arranged tightly into ductules, encapsulating the earlier portal area like sheath, and partly encapsulating the primary portal area. With the development of the secondary portal area, the oval like stem cells were gradually migrating to Hering canal. In addition, during various stages of the embryogenesis, there would be some mononuclear cells, which were positive for OV6 and CD34, and dispersing in the hepatic cords and sinusoids, especially in the mesenchymal tissue of portal area.Conclusion First, at the earlier stage of embryogenesis, the limiting plate, with abundant stem cells, perhaps is the origin of liver development. These stem cells are thought to have both clonogenic and bipotential capacity-that is, the ability to proliferate and differentiate into cells of either hepatocyte and biliary epithelial cell lineage. Second, the oval-like stem cells which expressed OV6, CD34, C-11 and CK19 respectively had the similiardistribution changes and morphological features, probably they are the same cells co-expressing hepatic stem cells' special surface protein. Thirdly, there also existed some mono-nuclear cells in fetal livers, that came from hematopoietic stem cells, which would give rise to a new intrahepatic stem cell lineage. The microenvironment in the liver induced these external stem cells to differentiate into a little part of the hepatic parenchymal cells.Part 2: Expression of Nestin in human fetal hepatic stem cells. Objective To explore the feasibility of inducting and differentiating the hepatic stem cells into insulin-secreting cells, we examined the expression of nestin in human fetal hepatic stem cells by immunohistochemical staining during various stages of the development. Meanwhile, we attempted to find a new specially expressed protein in these bipotent cells. Methods ABC staining was used to identify the special surface marker, nestin, in human fetal hepatic stem cells. Results The hepatic stem cells in the limiting plate were nestin positive. The unique cells arranged tightly in monolayer into ductules, encapsulating the earlier portal area like a sheath, and partly encapsulating the primary portal area. With the development of the secondary portal area, the oval-like stem cells were gradually migrating to Hering canal. In addition, during various stages of the embryogenesis, there would be some mononuclear cells, which expressed nestin that dispersed in the hepatic cords and sinusoids, especially in the mesenchymal tissue of portal area.Conclusion The hepatic stem cells which are believed to have both clonogenic and bipotential capacity were positive for neuroepithelial stem cell expressing protein nestin. Therefore, the hepatic stem cells have the potential to differentiate into islet hormone-producing cells. Meanwhile, there lived other source of cells which were also Nestin positive in the liver. Furt...