| Diabetes is one of the most common metabolic diseases in the world.The current treatments include islet transplantation and the use of hypoglycemic drugs,but the former lacks transplant donors and has the risk of transplant rejection,while the latter will have side-effects.Pluripotent stem cells can differentiate into insulin-secreting cells by directional induction,which may provide a new therapy for the treatment of diabetes.Human adipose mesenchymal stem cells?haMSCs?are derived from adipose tissue,easy to be isolated and cultured,and rarely involved in medical ethical issues,making them an ideal source of cell therapy for such as diabetes.MiR-375,Pdx1 and MafA play important roles in the development and maturation of pancreas and islet cells.As islet-specific miRNA,miR-375 mediates the post-transcriptional regulation of gene expression and can promote the differentiation of cells into insulin producing cells?IPCs?.Pdx1 is an important transcription factor in the development of the pancreas,regulating the insulin secretion in?cells of the pancreas and activating the expression of specific genes.MafA could induce the secretion of insulin in vitro under the synergistic effects of Pdx1 and other genes.In order to explore the effects of overexpression of these three transcription factors on the differentiation of haMSCs into IPCs,the miR-375,Pdx-1 and MafA expressing lentiviral vectors were used to transfect haMSC,and the transfected cells was used to differentiate into insulin-secreting cells.Firstly,the adipose-derived mesenchymal cells were isolated and cultured from the adipose tissue that excised from the donor's anterior abdominal wall.After in vitro culture and multiple passages,the cell morphology remained similar to that of fibroblasts,and keep the adherent growth.The osteogenesis,cartilage and adipogenic differentiation of haMSC were identified by Alizarin Red,Alcian Blue,Oil red O and BODIPY probe staining,respectively.The results showed that haMSCs have the ability to differentiate into bone,cartilage and fat cells.The expression of specific cell surface markers was analyzed by flow cytometry,the results showed that the haMSCs are highly expressing CD44,CD73,CD90,CD105,CD166 and other positive markers and negative for CD34,CD45,CD133,CD271.These results indicated that the haMSCs were successfully derived.Secondly,the lentiviral vector was constructed based on the gene sequences of miR-375,Pdx1 and MafA,and used for infection and construction of the miR-375,miR-375 and Pdx-1,miR-375,Pdx-1 and MafA overexpressing haMSC cell lines.These cells could identified by the expression of green fluorescent protein after 72hours of infection.The infection rate was about 80%,and the cells still expressed green fluorescent protein after passaging,indicating that the stable overexpressed cell lines were successfully constructed.Finally,the three-stage differentiation system was used for differentiation,which include 4 days culture in basal high glucose medium supplemented with Cyclopamine?CYC?,NOGGIN and Retinoic acid?RA?,3 days in a basic high glucose medium containing Nictinamide?Nic?,NOGGIN and Glucagon-like peptide1?GLP1?,and 7days in IMDM medium supplemented with Nic,NOGGIN,GLP1 and Insulin-like growth factors?IGF1?was cultured for.This system can successfully induce the differentiation of haMSCs into IPCs.The results of RT-PCR analysis of overexpressing cells differentiating into IPCs showed that the up-regulated expression of pancreatic related genes such as Insulin,Glucagon,Pdx1,Ngn3,Foxa2,Isl.1,and Nkx6.1 compared with those control cells,and the miR-375,Pdx-1 and MafA overexpressing cells showed higher levels of relative expression of Insulin,Pdx1 and Ngn3 compared with other groups,but Glucagon was up-regulated to a lesser extent.The results of immunofluorescence staining showed that the Insulin,Glucagon,Pdx1 and Somatostatin was positive in the cells.The Elisa assay showed that the average individual cells in the group miR-375,Pdx-1 and MafA exhibited insulin secretion of 3.6×10-4?g,while no secretion of insulin was detected in the other groups.This research indicates that overexpression of miR-375,Pdx-1 and MafA can promote the differentiation of haMSCs into IPCs,and miR-375,Pdx-1 and MafA overexpressing cells had better differentiation effects than those with miR-375,miR-375 and Pdx-1 overexpressing cells.It is suggested that overexpression of multiple pancreatic differentiation transcription factors can synergistically promote the differentiation of haMSCs into IPCs,which may provide a clinical study for the treatment of diabetes with autologous cells. |
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