The Primary Study On Induction Of Somatic Cells Into Insulin-secreting Cells

Diabetes is one of the major diseases that affect human health, which divided into type 1 and type 2. Patients with type 1 diabetes whoseβ-cell have the defects on insulin secretion require life-long treatment with exogenous insulin, some patients with type 2 diabetes also need to rely on insulin when taking the drug to lower blood glucose is beyond the control of blood glucose. In this case, the fundamental treatment is pancreas or islet cells transplantation, however, which is hindered by the limited availability of insulin-producing tissues and the subsequent need for life-long immunosuppression. Therefore, finding an unrestricted source of islet cells alternatives—cell-replacement therapy, has become the focus of research in rencent years. In this study, we selected the human embryonic kidney cell line (293T) and human embryonic fibroblasts (hEFs) for the target cells to induce insulin secretion.Objective: Transfect some important transcription factors relative to pancreaticβ-cell development into 293T and hEFs cells and express the transcription factors in cells utilizing genetic engineering method, so that the cells could become insulin-secreting cells by cells programming, then, we need to explore the molecular mechanism of these cells programming.Methods: After the open reading frames of Pdx-1, Ngn3, and Pax4 were amplified, these interest genes were integrated into adenovirus vector pAAV-MCS to construct recombinant vector. Then, these recombinant vectors were transfected into 293T and hEFs cells, respectively or two of them or three together. After 48h incubation, testing whether there is any insulin secretion in in 293T and hEFs cells from RNA level and protein level, and detecting the changes of isletβcell related genes.Results: When transfecting the recombinant plasmid Pdx-1 or Pdx-1 with other plasmids into 293T cells, insulin secretion was appeared 48 hours later. Selecting the combination ways that the maximum insulincontent secreted in 293T cells to deal with hEFs cells, insulin secretion was appeared in hEFs cell also. RT-PCR results showed that some pancreas related genes Ngn3, beta2 and pc2 which did not express in 293T and hEFs cells were appeared in transfected cells, in addition, immunofluorescence results showed that a certain amount of insulin secreted in transfected 293T cells, as well as a small amount of Glucagon, Amylin.Conclusions: Pdx-1 could promote cell programming and insulin secreting of 293T and hEFs cells.