The Effects Of Liraglutide On Differentiation Of Human Bone Marrow Mesenchymal Stem Cells Into Insulin-producing Cells

ObjectiveTo investigate whether human bone marrow mesenchymal stem cells(hBM-MSCs) could be differentiated into insulin-producing cells(IPCs) by liraglutide, and to find out the optimal concentration and time of this drug.Methods1. In vitro, P5 cells of HBM-MSCs were induced into IPCs by three-phase induction procedure. The details was that, hBM-MSCs were cultured in HG-DMEM medium containing 25mmol/L glucose for 10 days firstly. And then cells were transferred into LG-DMEM medium including 10mmol/L nicotinamide for 7 days. Finally cells were cultured in the LG-DMEM medium supplemented with different concentrations liraglutide (0.1, 1, 10, 20, 50nmol/L) for different days (1, 3, 5, 7, 9days).2. The morphological change of cells was observed by inverted microscope during induction. And the induced cells were confirmed by dithizone (DTZ) staining. The protein expression of PDX-1, GLUT2, GK and insulin in each induced phage cells was detected by western blot. And the insulin level and the response to high glucose were measured by ELISA.Results1. Liraglutide's optimum induction concentration and time was 10nmol/L and 7 days respectively in the study. And these results were confirmed by measuring the level of insulin in cultured supernatants with different concentrations (0.1, 1, 10, 20, 50nmol/L) and different induction days (1, 3, 5, 7, 9 days) of liraglutide.2. The morphology of hBM-MSCs was spindled, fibroblast-like before induction. However, cells began to aggregate and get round gradually in the first phase, and then the number of spherical cells increased, the cells distended in the second phase. In the last phase, the morphology of most cells was grape-like aggregation and islet-like cell clustered.3. The DTZ staining of hBM-MSCs was negative before induction. There were a few DTZ positive cells at the end of the first induction phase, and the positive rate was about 1%. The number of DTZ positive cells was increased in the second and third phase. And the positive rate was approximately 4% and 8% respectively.4. No protein expression of PDX-1, GLUT2, GK and insulin was detected before induction. The level of these proteins was increased gradually in each induction phase (P <0.05) .5. There was scarcely insulin detected in cell supernatant before induction. And the level of insulin was gradually increased after induction, and the cell response to high glucose was also increased gradually (P <0.05).Conclusions1. HBM-MSCs could be induced into IPCs by high glucose, nicotinamide and liraglutide respectively in vitro. And the inducing effect was pronounced after adding liraglutide of 10nmol/L for 7 days, the level of cell insulin secretion was obviously increased, and the response of the induced cells to high glucose was also significantly increased.2. The level of PDX-1, GLUT2, GK and insulin protein expression was significantly higher than that of two other stages, which confirmed that these induced cells were IPCs in protein level. These results may suggest that the increase of PDX-1 protein level could up-regulate the expression of many islet differentiation-related genes downsteam, such as, GLUT2 and GK. This illustrates liraglutide plays an important role in promoting hBM-MSCs to differentiate into IPCs in vitro.