Research Of Induced Pluripotent Stem Cells Directional Differentiated To Insulin Secreting Cells

Background:induced Pluripotent Stem Cell (iPS cell) is a kind of pluripotent stem cell with stem cell characteristics from somatic cells which is reprogrammed by exogenous factors. iPS cell has characteristics of self-renewal and pluripotency which is similar to embryonic stem cell. Studies have shown that human embryonic stem cell can differentiate into pancreatic beta cell and the expression of related specific markers, and it can be used to treat diabetic mice model successfully.Objective:To discover the possibility of human iPS cell differentiate into insulin secreting cells with the induction of related growing factors and to study expression and relationship of stem gene Oct4and beta cells related gene Pdxl, MafA and insulin.Method:iPS cell could be differentiated into insulin secreting cell by inducted of related growing factors by four stages. Expression changes of genes were detected by real-time quantitative PCR. Expression changes of related protein between iPS cell and differentiated cell were detected by Western-Blot. We also used cell immuno fluorescence technique to detect expression of related protein in differentiated cells. The glucose-stimulated C-peptide release experiment could be used to verify the successful progress of differentiation. Efficiency test was completed by flow cytometry.Results:Expression of stem factor Oct4decrease occurred mainly in the first two stage, there is no significant difference between stage three and four. During the process of differentiation, beta cell related factor Pdxl can be detected in stage one and keep increasing, expression of MafA is low in the first stage but it start to increase in stage three and four; insulin gene is detected at the terminal of stage two, but there is high expression in stage three and four. Results of Western-blot show the mature cells of differentiation have low expression on Oct4protein and high expression on Pdxl, MafA and insulin. Cell immuno fluorescence experiment shows that portions of these cells have successful expression of genes related to insulin secretion at the end of differentiation; the glucose-stimulated C-peptide release experiment shows in different concentration of glucose stimulation, differentiated cells secret C-peptide and there is significant difference(t=2.878, P<0.05),insulin secreting can be proved and the efficiency of differentiation is5.40%according to flow cytometry.Conclusion:iPS cell can differentiate into insulin secreting cells by inducted of related growing factors, during the process of differentiation, stem factor Oct4are decreasing, beta cell related factor Pdxl expressed and increased first, expression of Pdx1, MafA and insulin work together to promote the secretion of insulin and C peptide of differentiated cells.