| Objective: we set up STZ-injured INS-1cells,thenco-cultured them with bone marrow mesenchymal stem cells indirectlyby Transwell culture plate,and intervented with different concentrationof total saponins of panax ginsenoside(TSPG), to observe the effects ofTSPG on the bone marrow mesenchymal stem cells differentiation intoislet-like cells, and preliminary discuss the mechanism. Methods: wecultureed the INS-1cell and BM-MSC in vitro respectively, take the INS-1cell inoculated on transwell culture plate lower. when the cells growedup to80-90%confluence, synchronized the cells for24h with serum-freeculture, and treated them with different concentrations of STZ toestablished STZ-injured INS-1cells, then divided into5groups:(A)BM-MSC cultured alone group;(B) BM-MSC+INS-1co-culture;(C)BM-MSC+INS-1+25mg/L TSPG;(D) BM-MSC+INS-1+50mg/LTSPG;(E) BM-MSC+INS-1+100mg/L TSPG.Each group was culturedwith the same medium that consisted of50%1640culture medium，50%DMEM-L. The BM-MSC of B, C, D, E group were vaccination intranswell plate upper.9days later, identified the insulin-like cells withdithizone(DTZ) dyeing, and the5groups of cells were detected thePDX-1expression levels by Western-blot and detected the expression of insulin1and insulin2mRNA by RT-PCR. Results:(1) The cell modelof STZ-injured INS-1cells: INS-1cells were treated with differentconcentrations of STZ for24hours, the content of malondialdehyde(MDA) increased, and increased more significantly in the concentrationof0.8mg/ml,that showed a significant cell damage.（2）The insulin-likecells that differentiation from BM-MSC were identified withdithizone(DTZ)：the cells of BM-MSC cultured alone cannot be dyed, thecells of the rest of the groups were dyed into brownish yellow in differentdegree. That confirmed the existence of insulin-producing cells.(3) Theexpression of PDX-1protein in each group: the cells of BM-MSCcultured alone did not express the protein of the PDX-1, the cells ofco-culture groups were all expressed,and compared with the groupwithout TSPG, PDX-1protein were increased in TSPG groups(p＜0.05),and with the increase of TSPG, the protein expression of the PDX-1showed a trend of increase.（4） the insulin1and insulin2mRNAexpression in each group: the cells of BM-MSC cultured alone did notexpress insulin1and insulin2mRNA, the cells of co-culture groups allexpressed,and compared with the group without TSPG, insulin1andinsulin2mRNA were increased in TSPG groups(p＜0.05), and with theincrease of TSPG, the expression of the insulin1and insulin2mRNAshowed a trend of increase.(5) Glucose stimulation experiments: culturethe insulin-like cells with5.6mmol/L and16.7mmol/L glucose medium for2hours respectively, insulin excreted from differentiated cells wastested with enzyme-labeled immunosorbernt assay (ELISA). No insulinwas detected in the BM-MSC cultured alone group,while the cells ofco-culture groups secreted more insulin at16.7mmol/L than5.6mmol/L(p＜0.05). Conclusions:(1)Treated the INS-1cells with STZcan cause oxidative damage in vitro.(2)BM-MSC co-cultured with INS-1cells indirectly could be induced into islet-like cells, and the islet-likecells could secrete insulin in the stimulation of glucuse.(3) TSPG have apromoting effect during the process of BM-MSC differentiation intoislet-like cells，and have concentration-dependent manner in vitrowithin a certain range.This effect may be achieved by the up-regulatingeffect on protein expression of PDX-1and mRNA expression of insulin. |
