Tracing For Hepatocyte-like Cells That Difierentiated From Human Peripheral Blood Mononuclear Cells Labeled By Me Fluorescent Dye PKH26after Transplantation In Nude Mice

Part OneHuman peripheral blood mononuclear cells induced differentiation into hepatocyte-like cells by hepatocyte growth factor and fibroblast growth factor-4in vitroObjective:To induce human peripheral blood mononuclear cells differentiate into hepatocyte-like cells by hepatocyte growth factor (HGF) and fibroblast growth factor-4(FGF-4) in vitro. To observe and detect whether the differentiated cells with the typical morphological and biochemical characteristics of hepatocyte-like cells.Methods:Density gradient centrifugation and cell adherent screening isolated and purified the mononuclear cells from peripheral blood to culture and induce. Group A and B were set up respectively in experiment. In Group A, mononuclear cells were cultivated without hepatocyte growth factor (HGF) and fibroblast growth factor-4(FGF-4) in cell culture. They were used as negative control group. In Group B, mononuclear cells were cultured with the administration of both hepatocyte growth factor (HGF) and fibroblast growth factor-4(FGF-4) to induce the differentiation into hepatocyte-like cells. The changes in cell morphology were observed and the expressions of AFP and CK19were detected by immunocytochemical staining in two groups at different times after induction.Results:Cells in group A that spherical shape was observed when peripheral blood mononuclear cells were just isolated are gradually becoming inconformity in morphology, to present spindle or fibroid, and a few cells are round, the cells developed apoptosis and cracked following incubation. Cells in group B have a strong proliferative activity. They become large and oval, grows in colonies following induction. They have typical hepatocyte-like cell morphology after two weeks of induction. The expressions of AFP and CK19that detected by immunocytochemical staining were negative in group A after incubation. Inversely, the expressions of AFP and CK19that detected by immunocytochemical staining were positive in group B after induction. The positive expression of AFP and CK19in group B was most obvious after two weeks of induction.Conclusion:Human peripheral blood mononuclear cells have the potential to differentiate into hepatocyte-like cells under the induction of hepatocyte growth factor and fibroblast growth factor-4and the differentiated cells have the typical morphological and biochemical characteristics of hepatocyte-like cells. Part TwoTracing for hepatocyte-like cells that differentiated from human peripheral blood mononuclear cells labeled by the Fluorescent Dye PKH26after transplantation in nude miceObjective:To determine whether PKH26could be used to serve as an effective tracer for the hepatocyte-like cells differentiated from human peripheral blood mononuclear cells, and observe the ability of transplanted hepatocyte-like cells differentiate into hepatic cells in nude mice.Methods:The hepatocyte-like cells that differentiated from human peripheral blood mononuclear cells labeled by the fluorescent dye PKH26and injected via caudal vein into nude mice as experimental group. The nude mice injected the equal amount of normal saline in experimental control group. The labeled cells of the status of the migratation and the outcome are observed by the fluorescence microscope in the hepatic tissue sections of nude mice and the expressions of albumin (ALB) were detected by immunocytochemical staining after two weeks the cells transplantation.Results:The hepatic tissue sections of nude mice in the experimental group showed numerous the PKH26labeled cells. The hepatocyte-like cells that labeled by PKH26distributed mainly in the hepatic cord area, integrated in hepatocyte and expressed ALB in the liver biopsies of the nude mice after two weeks of transplantation. The experimental control group did not observed the PKH26labeled cells in the hepatic tissue sections of nude mice.Conclusion:PKH26can be an effective tracer for hepatocyte-like cell transplantation. The hepatocyte-like cells in hepatic tissue migrated from the hepatic sinusoidal area to the hepatic cord area, and implanted, integrated in hepatocyte and differentiate into mature hepatocyte to express ALB after transplantation.