Human Bone Marrow Mesenchymal Stem Cells Were Differentiated Into Insulin-secreting Cells By Transfection With PDX-1 Gene

Objective:To study the multitude potency of differentiating into osteoblast cells by oriented induction of bone marrow mesenchymal stem cells(MSCs).PDX-1 gene transfection induced human bone MSCs into insulin secreting cells by use of gene modified,and explore the value of Bone MSCs in the field of diabetes treatment.Methods:1.MSCs were isolated with Percoll density gradient centrifugation of blood from adult bone marrow.The 3rd generation cells were identificated with immunofluorescence methods such as CD44 antibody,CD45 antibody,CD34 antibody. Observe biological properties of the cell with growth curve and electron microscopy.2. The 3rd generation cell were induced into osteoblast using containing dexamethasone,β-glycerophosphate,vitaminC,observe morphological changes of MSCs with alkaline phosphatase(ALP) staining,the group was not induced as a parallel control group.3.The PDX-1 gene recombinant vector PEGFP-N1- PD-X-1 was constructed by way of RT-polymerase chain reaction,and which were transfected into human bone MSCs.The expression of PDX-1 protein was detected by immunofluorescence;suspended cells were identificated by dithizone staining,insulin which was secreted by genetically modified cells was detected electricity chemiluminescence.Result:PDX-1 gene was inserted correctly into the recombinant plasmid by way of gene sequence assay,PCR and restriction enzyme digestion.Green fluorescent protein could be found in human bone marrow mesenchymal stem cells which proved that human MSCs were transfected effectively through the fluorescence microscope observation.PDX-1 protein expressed positive one week later.Suspended islet like cell clusters could be seen in human bone marrow MSCs after two weeks,and dithizone staining positive.Insulin capacity which was secreted by transfected cells was determined through electrochemiluminescence in the high-sugar and low-sugar group.Conclusion:PDX-1 gene recombinant vector were successfully constructed,which could be transfectd into human bone marrow MSCs,expressed stably,and differentiated into insulin-secreting cells.The experimental basis concerning the expression of PDX-1 gene in bone marrow MSCs as seed cells for tissue engineering was provided.