Construction Of The PDX-1Cloning Vector In Vitro And Inducing The Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells Into Insulin Secreting Cells In Vitro

Objective: To build the PDX-1gene cloning vector for constructing eukaryoticexpression vector and studying the mechanisms of stem cell differentiation induced byPDX-1.Methods: RNA was extracted from insulinoma cell line of rat by Trizol method, PDX-1cDNA was amplified by RT-PCR method in vitro, nucleoside sequence was identifiedby agarose gel electrophoresis, and then the fragment was inserted into pMD-18Tcloning vector. The recombinant cloning vector pMD-18T-PDX-1was identified bydouble digestion and DNA sequence analysis.Results: The recombinant cloning vector pMD-18T-PDX-1was constructed in vitro,and the length of PDX-1cDNA was908bp.Conclusion: The PDX-1gene was successfully amplified in vitro, and the recombinantPDX-1cloning vector was successfully constructed. It is convenient for expression ofPDX-1in eukaryotic cells and studying the role of PDX-1in the directed differentiationof stem cells. Objective: Human umbilical cord mesenchymal stem cells were cultured and inducedinto insulin secreting cells differentiation in vitro. To explore the possibility andfunctional trends of inducing the differentiation of stem cells into insulin secreting cells.Methods: Human umbilical cord mesenchymal stem cells were cultured to adherenteighty percent, and then mesenchymal stem cells were divided into two groups: group Afor the induced group and group B for the control group. Group A was induced by1mmol/L thioglycol2-mercapitoethanol for two days,10ng/ml epidermal growth factor,10ng/ml basic fibroblast growth factor and two percent B27for seven days. At lastgroup A was induced by20mmol/L nicotinamide and exenatide for seven days. GroupB wasn’t induced. The morphologic changes of insulin secreting cells were investigatedby invert microscope; the expression of PDX-1gene was amplified by RT-PCR and thesecretion level of insulin was detected by radioimmunoassay.Result:(1) The undifferentiated umbilical cord mesenchymal stem cells changed fromfusiform to fibroblast-like growth and had clear boundaries.The cells of group A wereround in shape and became smaller and smaller, some of them aggregated into groupsand were similar to the islet-like cells. But the morphology of group B wasn’t changed.(2) The PDX-1gene from group A could be amplified by RT-PCR, and the length ofPDX-1cDNA was912bp complied with pubmed.The PDX-1gene from group B couldnot be amplified by RT-PCR.(3) The insulin secretion level of the two groups wasdetected by radioimmunoassay. Group A could be detected but group B could not bedetected. Conclusion: Human umbilical cord mesenchymal stem cells were induced into insulinsecreting cells successfully in vitro, and it provide a basis for the application of stemcells in the field of diabetes treatment.