Study On The Small Molecule Compounds Allow The Reprogramming Of Mice Hepatocytes Into Insulin-producing Cells

Objective: To establish the technical system of small molecule compounds in vitro reprogramming of mice hepatocytes into insulin-producing cells,we use the small molecule compounds reprogram mice hepatocytes into insulin-producing cells phasedly in vitro.The new technical system could help to further explore the mechanisms and signaling pathways of the hepatocytes conversion into insulin-producing cells and lay the foundation of efficient transdifferentiation the hepatocytes into insulin-producing cells.Methods:1.We adopt the method of direct shearing the liver tissues of the mice to obtain the primary hepatocytes and culture them.2.Small molecule compounds,such as 5-aza-2?-deoxycytidine,trichostatin A,insulin-transferrin selenium,retinoic acid and nicotinamide allow the reprogramming of mice hepatocytes into insulin-producing cells in three steps.3.We use hepatic glycogen staining to identify of primary hepatocytes,real-time quantitative PCR and immunofluorescence to detect the gene and protein expression levels of the stages of the cells,insulin release experiments and mouse ELISA to assay IPCs’ function,flow cytometry to detect the efficiency of small molecule compounds reprogramming hepatocytes into insulin-producing cells.Results:1.According to hepatic glycogen staining and flow cytometry,we find that most cells are glycogen granules positive and the positive rate of ALB is 86.2%after cultured 48 h of the primary cells obtained from directly shreded mice liver tissues.2.Hepatocytes treated with 5-AZA two days and TSA one day reach a state of dedifferentiation,which show the expression of hepatocyte’ marker ALB significantly reduces and the early pancreatic development related gene Sox9 and nestin are positive.The dedifferentiated cells differentiate into pancreatic progenitor cells,namely Pdx1 positive cells,after treated with RA and ITS seven days.Pancreatic progenitor cells further differentiate into IPCs under the influence of NA.3.The insulin release experiments show that the pancreatic precursor cells have no response to different concentrations of glucose stimulation,while the insulin secretion of IPCs are different to different concentrations of glucose stimulation.The result of flow cytometry shows that the rate of insulin-positive cells in the third stage is 2.6% during the IPCs reprogramming process.Conclusion: After treated with small molecule compounds 5-AZA and TSA,mice hepatocytes reach a brief dedifferentiation state.Futher under the action of retinoic acid,ITS and nicotinamide which can be converted into the functional insulin-producing cells.