Study On Differentiation Of Liver Stem Cells Into Pancreatic β Cells

Recently, Diabetes becomes one of the serious diseases that affect human health. It can be divided into typeⅠand typeⅡdiabetes according to the absolutely or relatively lack of the insulin secretion. TypeⅠdiabetes (T1D) is a disorder characterized by the autoimmune-mediated destruction of insulin-producingβcells in the pancreas. Insulin replacement and drug therapy for T1D could relatively maintain euglycemia, but they have proven to be ineffective in preventing long-term complications associated with fluctuations in blood glucose levels. Moreover, pancreas and islet cell transplantation, while effective in achieving insulin independent, persistent euglycemia, is limited by the requirement of life-long immunosuppressive medication and the lack of donor pancreata. Especially, the latter seriously hampered this approach application in clinic. With the further study in stem cells differentiation and differentiated cells transdifferentiation, the strategy that differentiate/transdifferentiate non-β-cells to insulin producing cells to cure diabetes becomes research hotspot.Previous studies demonstrated that transdifferentiation/reprogramming of cells sharing a common embryonal origin can often be achieved by expressing tissue-specific/selective transcription factors (TFs) from one cell type into the other. In embryonic development, liver and pancreas develop from the ventral foregut endoderm at a similar time. The developmental homology decides that there is the similarity in controlling the cell proliferation and differentiation in the liver and pancreas. Thus, these cells from the liver could be easily transdifferentiated to pancreaticβcells. Recent many studies demonstrate that it is relatively easy to conversion or transdifferentiation of hepatic stem cells, human fetal liver cells, human hepatoma cells and human adult hepatocytes into functional insulin producing cells by ectopic expression of key pancreatic developmental transcription factors such as Pdx1, Pax4, NeuroD and MafA. The achievements in this area, on the one hand, expanded the sources ofβcells used to transplant to cure diabetes. On the other hand, they provide a new strategy to set up the insulin producing cells in diabetic patients'liver to achieve the therapeutic purposes.However, there has not a technical system which can be used to transdifferentiate liver cells to insulin-secreting cells specifically and efficiently. In this study, the full length of Pdx1,MafA,NeuroD,Pax4 genes was separately amplified by PCR and subcloned into pAd-Track-CMV shuttle vector plasmid. Then the recombinant adenovirus plasmids were generated in AdEasier-1 cells. The recombinant adenovirus were generated in the packaging 293LP cells. After 48 hours separately infected with adenovirus at multiplicity of infection (M.O.I) of 20, the human fetal liver stem cells (hFLSCs) almost expressed the green fluorescence protein (GFP) report gene. Then the transcription and the translation of objective gene were detected by RT-PCR and Western Blot separately. After injected adenovirus via caudal vein, nearly all of the mice liver cells were detected the expression of the GFP report gene six days later. The results demonstrated that the adenovirus vectors expressing this key pancreatic transcription factors were constructed successfully. It lays a good foundation for studying the transdifferentiation of liver stem cells to pancreas lineage cells and the establishment of insulin producing cells in mice liver in vivo. To establish the technical system that could be used to induce the liver stem cell to differentiate to pancreaticβcells specifically and efficiently, LEPCs were affected at total M.O.I of 20 with Pdx1,MafA,Pax4 and NeuroD separately or combinantly. The results indicated that some of LEPCs expressed the insulin 1 and insulin 2 genes after affected with Pdx1 and MafA adenovirus three days. This finds lay the foundation for further study to differentiate the liver stem cells to pancreaticβcells.