| Liver failure is the most common clinical liver syndrome with high mortality,which is a serious threat to human health.According to statistics, there are 30-50 million people dead because of severe liver failure every year.Liver transplantation is the most effective way to treat liver failure, however,only less than 30%of patients have the opportunity to receive liver transplantation because of donor source scarcity.In recent years,It has been found that liver stem cell transplantation has effectively therapeutic effect on acute and chronic liver failure.Hepatic stem cells (HSC) have the characteristic of self-renewal and multipotential differentiation,which can be differentiated into hepatocytes,bile duct epithelial cells and other cells.In liver injury,liver stem cells may be involved in liver repair and regeneration.HSCs can differentiate into mature functional hepatocytes,serving as a major cell source of liver cell transplantation and bioartificial liver cells.Embryonic stem cells, haemopoietic stem cell,bone marrow-derived mesenchymal stem cells, embryonic liver stem cells,hepatic oval cells can be used in live stem cell transplantation.Although there have been numerous reports indicating that different sources of liver stem cells can be differentiated in functional hepatocytes in vitro and in vivo,the differentiation efficiency was relatively low.How to achieve terminal differentiation and fully functional hepatocytes remains as a major challenge.Whether the transplanted cells can proliferate normally and replace liver mass functionally is still unknown.It has been reported that the up-regulation of HSC related genes could improve the efficiency of HSC transplantation in the treatment.Embryonic liver stem cells(ELSC) are the progenitor cells,which can only differentiate into hepatocytes and bile duct epithelial cells.The intermediate state is an essential process of differentiation from stem cells to mature hepatocytes.While investigations on embryonic liver stem cells are helpful to elucidate the mechanism of liver development,the directed differentiation of liver stem cells into mature liver cells,but not tumor cells, would significantly improve the efficiency and biosafety profile of clinical use of liver stem cell transplantation.During liver development,Wnt signaling is temporarily activated, triggering a complicated signaling cascade that regulates cell proliferation, differentiation,growth,migration,development and oxidative stress.Wnt signaling plays important roles in liver development and liver cell differentiation.Current studies on the role of Wnt signaling pathway in the liver development and differentiation mainly focus onβ-catenin,while the individual members of Wnt family and Wnt antagonists have been poorly studied.Furthermore,the expression profiles of all Wnts during liver development has not been studied.Thus,we are interested in studying the functional role of Wnt signaling and its antagonist in liver stem cell differentiation in the following three parts.Part One Expression Profiles of Wnt Signaling Molecules in Different Developmental Stages of Liver Tissues and Cell LinesObjective:To detect the expression levels of all Wnt ligands, receptors and antagonists in different developmental stages of liver tissues and stem cell lines,then to analyze the expression of differences and trends, for our further study on the role of Wnt signaling pathway in the liver development and cell differentiation.Methods:Liver tissues were isolated from 12.5 embryos to postnatal 4 weeks mouse liver,mouse embryonic liver stem cell 14-19 and liver cell 14d were isolated and established from post coitus day 14.5 and postnatal 14 days liver tissues,respectively.The expression profiles and trends of 19 Wnt ligands,10 Fzd receptors,two co-receptors and 8 antagonists were detected by RT-PCR.Results:Most of Wnt genes were detectable.Among them,Wnt2b,3a, 4,8a,9b,10a,and 10b were highly expressed at all of the tested stages of liver development.It is noteworthy that Wnt2 and 5a seemingly expressed at the later stages,while Wnt6 and 16 expressed at the early stage of liver development.All ten Fzd receptors were expressed in the isolated liver tissues,while the expression of Fzd2,3,5,6,7,and 8 slightly decreased during liver development.Fzd4 and 10 seemingly increased their expression postnatally.The expression of both LRP5 and LRP6 was readily detected in all liver tissues,although the expression of LRP6 increased after birth.SFRP1,4 and 5 were highly expressed during liver development. However,SFRP2 only expressed in E12.5 liver tissue,whereas the expression of SFRP3/FrzB was only detected in E12.5 through E14.5 liver tissues Accordingly,Dkk1 and Dkk3 were shown to express in all of the isolated liver tissues,whereas Dkk2 mostly expressed in E12.5 liver tissue.The expression of 9 of 19 Wnt genes was readily detected in ELSC14-19.In Hepa1-6,Wnt1,10a had higher expression,Wnt2 disappeared and Wnt3a,6 presented.Most of the 10 Fzd receptors and LRP5,6 co-receptors were highly expressed in ELSC14-19.Among the five SFRP antagonists,SFRP2 and 4 were highly expressed,SFRP1 and 5 lowerly expressed,while the expression of Frzb was almost not detectable in ELSC14-19.Dkk2 exhibited the highest level of expression in ELSC14-19,whereas Dkk1 was expressed at the lowest level and Dkk3 expression was readily detectable.The expression of SFRP2,DKK2 were higher in ELSC 14-19 than in LC14d,but SFRP4 lower.Furthermore,the expression of four known Wnt targets,c-Myc,Axin2,Sox9,and Nanog was readily detected in ELSC14-19 cells.Conclusion:Most of components of Wnt signaling pathway in the liver development were activated with different expression profiles,the trends of their expression in liver tissues and stem cells were not totally similar,while Wnts differentially expressed in the normal liver cell and liver tumor cell,suggesting that Wnt family members in differentiation of the liver may play different roles.Among them,Wnt antagonists SFRP2, Frzb,DKK3 only expressed in early stage of liver tissue,indicating that the down-regulation of them may be necessary in liver development and cell differentiation.Part Two Induced Differentiation of Embryonic Liver Stem Cell in vitroObjective:To construct ELSC14-19 stable cell line with ALB promoter and Gaussia luciferase reporter gene,for dynamic monitoring of differentiation status and factor screening.To detect the effect of different factors on ELSC induced differentiated in vitro.Methods:ALB promoter and Gaussia luciferase reporter gene were constructed in a retrovirus vector,then transfected ELSC14-19,LC14d,and Hepa1-6.Relative ALB expression level was detected by culture supernatant luciferase activity.Retrovirus was packaged in HEK293 cells with pAmpho co-transfection.ELSC14-19 ALB-GLuc stable cells pool was established by retrovirus infection.The effects of 10%FBS,2%HS, Dex,HGF,FGF4 single factors and different combinations of culture conditions on differentiation of ELSC14-19 in vitro were detected by ALB-GLuc activity assay.The effect of 2%HS + Dex + HGF + FGF4 combinations on differentiation of ELSC14-19 was also assessed by semi-quantitative RT-PCR,immunofluorescence,glycogen staining,and ICG uptake and release test.Results:Gaussia Luciferase activity reflected ALB expression.Cells proliferation slowed in 2%HS than that in 10%FBS culture condition,but ALB-GLuc activity higher.HGF,FGF4 at 10ng/ml,20ng/ml,respectively, could most effectively promote ALB expression.FGF4 improved cell proliferation.Compare with different combination,2%HS+0.1uMDex+ 10ng/ml HGF+20ng/ml FGF4 co-culture induced highest ALB expression, After inducted,the expression of DLK,AFP,CK19 decreased,ALB,CK18, UGT1A increased,and mature marker TAT was detected at 3 days of induction,and ApoB,at 6 days induction.ICG uptake and glycogen synthesis function of induced cells were present at 6 days induction,and gradually increased.Conclusion:We successfully constructed stable ELSC with ALB promoter-driven luciferase reporter gene.ALB-GLuc activity correlated the expression level of ALB.ELSC differentiation could be effectively induced in vitro by 2%HS+0.1uM Dex+10ng/ml HGF+20ng/mlFGF4.Part Three Effect of Wnt Antagonists on Embryonic Liver Stem Cell DifferentiationObjective:To detect and compare the effects and mechanisms of Wnt antagonists DKK2 and Frzb on embryonic liver stem cells in vitro differentiation.Methods:ELSC14-19 cells were infected with Ad-DKK2,Ad-Frzb, respectively,and cultured in 2%HS+0.1uM Dex+10ng/ml HGF+ 20ng/ml FGF4 condition.Hepatic related markers were detected by RT-PCR,Western Blot at 12 days induction.The mature functions of ELSC were assessed by ICG uptake and glycogen synthesis tests. ELSC14-19 cells were transfected with pTop-Luc plasmid,then infected with adenovirus exprssingβ-catenin,19 Wnt ligands,DKK2,Frzb in different combinations.β-Catenin activity was detected by Luc-activity.Results:The expression of DLK,AFP,CK19 was higher in Adv-Frzb infected group than the control group.The expression of ALB,CK18, UGT1A,TAT,ApoB decreased after Frzb treatment.The ICG uptake and glycogen synthesis apparently reduced after Frzb treatment.However,there was no significantly difference between DKK2 treated induced group and induced control group.The Top-Luc activity(reflectingβ-catenin/TCF activity) ofβ-catenin,Wnt1,2,3,3a,7a,7b,10b treated groups were statistically higher than that in GFP control group.FrzB inhibitedβ-catenin, Wnt1,2.3,3a,7a,7b induced Top-Luc activity,and Luc activity induced by Wnt10b was not affected by FrzB.DKK2 inhibitedβ-catenin,Wnt1,2, 3,3a,7b induced Top-Luc activity,weakly improved Wnt10b induced Top-Luc activity,Luc-activity induced by Wnt7a was not affected by DKK2.Overall,the inhibition of Frzb on Wnt inducedβ-catenin activity was stronger than that of DKK2.Conclusion:Frzb could inhibit the differentiation induced by 2%HS +Dex+HGF+FGF4 co-culture,while DKK2 had no effect.These findings suggest that the inhibition of Frzb and DKK2 on Wnt signaling pathway in ELSCs may be different. |
