The Direct Oct4-mediated Primary Liver Cells In Mice Reprogramming Of Pdx-1Positive Cells

Objective Diabetes using traditional treatment methods can not avoid theoccurrence of long-term complications and blood sugar disorders, so it is necessaryfunctional islet beta cell replacement therapy. In2006, the mountains stretchindemnity and other screening four specific transcription factors (Oct4, Sox2, c-Mycand Klf4) import body cells induced have embryonic stem cell-like characteristics andfunction of multi-competent cells (iPS), islet β cell replacement therapy provides anew way.This experiment the lentivirus transferred by over-expression of Oct4gene,mediated mouse liver cell dedifferentiation Sox17-positive cells so that the latter, andfurther differentiation of Pdx-1positive cells binding small molecule compounds,exploring new ways of re-programming of the liver and pancreasMethods Cloning method to build the the lentiviral vector pWPT-Oct4plasmidby restriction enzyme digestion and sequencing, the use of lentiviral packagingsystems, packaging and concentrated virus; improved the classic two-step collagenaseperfusion via the hepatic portal vein were isolated mouse primary hepatocytesRT-PCRdetection of liver cells and endoderm transcription factor expression and activityidentification with liver cell culture medium; multiplicity of infection (MOI) of30mice primary hepatocytes infected with Oct4-lentiviral concentrate;Results On pWPT-of Oct4slow virus plasmid by restriction enzyme digestion,sequencing proved to be successful to build containing the Oct4lentiviral expressionvector. Liver cells in after lentivirus secondary infection, expression of the exogenousOct4confirmed Oct4has been successfully transferred to the liver cells; With thestable expression of Oct4gene in hepatocytes, the mature hepatocytes transcriptionfactor (Alb, Ttr AFP) expression gradually decreased, confirmed liver celldedifferentiation, further observed that the key genes in the early endoderm markersSox17and pancreas development process pancreatic duodenal homeobox iso-boxprotein-1(pancreatic duodenal homeobox-1, Pdx-1) expression in the control group did not appear at the same time without the pluripotency genes Oct4, Nanog and Sox2expression of endogenousConclusion Using lentiviral vector over-expressing Oct4, so that the lattermediated liver cell dedifferentiation to Sox17and Pdx-1expression in cells infectedlay the foundation for the further Pdx-1positive cells into insulin-producing cells.