The Study Of The Differentiation Of Rabbit Bone Marrowmesenchymal Stem Cells Into Into Islet Cells

【Objective】The experiment intends to explore an efficient induction system of directional differentiation of rabbit bone marrow mesenchymal stem cells into islet beta cells in vitro,thus it can provide a theoretical basis for the further research and clinical application of stem cells in the treatment of diabetes.【Methods】BMSCs were obtained by red blood cell lysis and whole bone marrow culture. The cell morphology of BMSCs was observed under inverted microscope, the cell viability was detected by trypan blue staining, the first time of passage was recorded, the CD90、CD44、CD34 antigen expression were detected by immunocytochemistry technique. The first time was changed respectively at 12h、24h、48h、72h, then all groups were exchange every 2d. The period of each passage were measured; This test we use P3 generation cells, growth factors three-step strategy was adopted to induce BMSCs to differentiate toward islet cells. The first phase contains 10 ng/m Lb FGF, 1% DMSO and 1% FBS, 23 tendency LGlucose H-DMEM culture of BMSCs induced by 3 d, the second phase containing 10 ng/m Lb FGF, 10 ng/m LEGF, 2% B27 and tendency for 17.5 / LGlucose, 10 tendency of nicotinamide serum-free DMEM/L F- 12 culture of BMSCs induced 8 d, the third stage with contain 10 tendency LHEPES, 10 tendency for L niacinamide, 2 nmol/L of activin A, the tendency for 11.1 / LGlucose RPMI1640 culture of rabbit BMSCs induced 8 d.The cell morphology of BMSCs was observed under inverted microscope;The differentiated cells were tested by dithizone staining(DTZ).RT-PCR were carried out to dectect the express of inslet cells related gene(Pdx-1、Insulin、Nkx6.1), and the expression of specific protein like Insulin in derived cells was investigated by immunocytochemistry technique; Insulin secretion was assayed by ELISA.【Result】 The result showed that BMSCs could be isolated by two methods. The adhere cells showed shuttle or gathered into a vortex-like uniform growth, and positive for CD90、CD44 antigen, negative for CD34. BMSCs isolated by the red blood cell lysis were superior to the whole bone marrow culture by the cells viability and primary culture time, the significant difference(p<0.05); 24 h in liquid time needed for each generation to extend the time for the first time the extremely significant difference compared with other groups(P < 0.05), as the best time of the liquid for the first time, can significantly reduce the cycle of the cell culture; At the end of the first-step strategy, the cell morphology had no obvious change, and part of cells become round, DTZ staining negative, any specific gene m RNA expression was detected. After inducted 7 days, BMSCs gradually merged to form cluster, DTZ staining positive, only PDX-1 m RNA expression was observed, which was verified as insulin progenitor cells.At the end of second-step strategy, the number of differentiated cells cluster was incteased, RT-PCR showed that Pdx-1 and Insulin were expressed. After inducted 19 days, cell clusters look like islet-like structure in terminal, DTZ staining positive and detected Pdx-1、Insulin、Nkx6.1 expression. Immunocytocheemistry analysis showed expression of PDX-1 in the islet-like clusters.The insulin concentrations of BMSCs group and induced group were(27.86±0.034 μIU/m L)、(29.33±0.48 μIU/m L)、(29.92±0.46 μIU/m L)、(31.32±0.03 μIU/m L)and 20.54±0.21 μIU/m L)、(22.10±0.45 μIU/m L)、(22.36±0.35 μIU/m L)、(23.39±0.20 μIU/m L)at the 3rd、7th、11th、15th、19th days, and the difference was very significant(P<0.05) between BMSCs and induced groups except the 3rddays.【Conclusion】Show that the red blood cell lysis method, 24 h for the first time in liquid can improve the activity of bone marrow mesenchymal stem cells,shorten the cycle of cell culture; It illustrated that isolated and purified BMSCs in vitro can be inducted to differentiate into fuctional mature islet cells.