人脐带间充质干细胞分化胰岛样细胞过程中胰岛素和巢蛋白的表达

Changes of insulin and nestin expression during the differentiation of human umbilical cord mesenchymal stem cells into islet-like cells

BACKGROUND:Domestic and international studies have confirmed that human umbilical cord mesenchymal stem cells could be induced to differentiate into islet-like cells, but little is reported about the changes of insulin and nestin expressions during the differentiation phase.OBJECTIVE:To observe the changes of insulin and nestin expressions during the differentiation of human umbilical cord mesenchymal stem cells into islet-like cells.METHODS:Human umbilical cord mesenchymal stem cells were cultured using UltraCULTURE medium in vitro. Stem cells were cultured for three generations to observe cell morphological changes under an inverted microscope, to test immunophenotype by flow cytometry, and to identify the capacity of osteogenesis and adipogenic differentiation. Induction protocol was divided into two stages. In stage 1, stem cells were induced for 14 days in the UltraCULTURE medium with 4 nmol/L activin A, 25 μg/L epidermal growth factor, 100 μg/L β-nerve growth factor, 10 mmol/L nicotinamide. In stage 2, the cells were cultured in the UltraCULTURE medium with 1% insulin-transferin-selenium, 10 mmol/L nicotinamide, 10 μg/L basic fibroblast growth factor for an additional 14 days. The expressions of nestin and insulin in those differentiated cells were tested by flow cytometry, and zinc ion expression in the islet-like cell clusters was identified by dithizone staining. RESULTS AND CONCLUSION:During the differentiation process, the insulin level was increased gradually in the induction group and reached a higher level on day 28, but the insulin expression showed negative in the control group. In addition, on day 14 of induced differentiation, the nestin expression reached the peak and then gradually reduced along with the prolonged inductive time. On day 28 of induction, islet-like cell clusters formed and were positive for dithizone staining. In this experiment, the umbilical cord mesenchymal stem cells were successfully induced and differentiated into islet-like cells, accompanied with the variation of insulin and nestin expression.