Impact Of Alcohol On Differentiation Human Emryonic Stem Cells Towards Hepatocytes

The study of embryonic stem cells(ESCs)is one of the hot fields in life science research.ESCs are used as cell models for liver disease research to solve problems such as slow growth of human liver stem cells,difficulties of the establishment of liver stem cells and the differentiation of liver stem cells to hepatocytes.Liver is the major organ responsible for ethanol metabolism and excessive drinking is the primary cause of severe liver diseases.In European countries and USA,alcoholic liver disease is one of leading causes of death in young people due to excessive drinking.The differentiation model of ESCs to liver cells was used in this thesis.In order to explore the effect of alcohol on liver cell differentiation,ESCs were induced to differentiate into endodermal cells first and then to differentiate to hepatocytes.By simulating the regeneration process of hepatic oval cells after liver injury,we intended to investigate the molecular mechanism for alcohol's effects on the differentiation of liver stem cells.This research found that alcohol inhibited the differentiation of human ESCs to hepatocytes and biliary cells as well as the formation of mature biliary cells,Meanwhile,alcohol suppressed the expression of liver metabolic enzymes and the establishment of metabolic functions.This study proved that alcohol could inhibit the ERK signaling pathway in the process of hepatocytes differentiation,decrease the phosphorylation level of GSK-3? phosphorylation site Ser 9,reduce the accumulation of ?-catenin in the nucleus,and down-regulate the expression of its downstream target gene Cyclin D1,and ultimately result in cell cycle arrest.During hepatocytes differentiation,adding U0126,the ERK signaling pathway inhibitor,suppressed the formation of hepatocytes and biliary cells.This phenomenon coincided with the process of alcohol's inhibition on induced differentiation.The combination of U0126 with the growth factors HGF and FGF4 made hepatocytes differentiation become normal and p-ERK and Cyclin D1 protein levels become normal.This confirmed that alcohol inhibited the effects of the growth factors HGF and FGF4 in activating the ERK signaling pathway,thus inhibiting the induced differentiation of human ESCs into hepatocytes.Furthermore,alcohol affected Wnt classic signaling pathway and Notch signaling pathway during hepatocytes differentiation,decreased the activities of these two signaling pathways,and suppressed the expression of WNT1.We added Notch pathway inhibitor during induced differentiation process,which inhibited the formation of biliary cells;the differentiation of hepatocytes was promoted by up-regulating Wnt1.Subsequently,we added IWR-1-endo,a Wnt classic signaling pathway inhibitor,which suppressed the formation of hepatocytes and promoted the formation of biliary cells;it also inhibited the expression of liver metabolic enzymes and the establishment of metabolic functions.These results demonstrated that in the induction system,Wnt classic signaling pathway controlled the formation of hepatocytes while Notch signaling pathway controlled the formation of biliary cells.The results of the study confirmed the effects of alcohol on the differentiation of human ESCs to hepatocytes and deepened our understanding on mechanisms of hepatocytes differentiation.The findings will help in guiding clinical studies on the treatment of liver injury and alcoholic liver disease in the future.