【作者】 毕杨；

【导师】 冯涛； 何通川；

【作者基本信息】 重庆医科大学 ， 生物医学工程， 2009， 博士

【摘要】 肝功能衰竭(Liver failure)是临床最常见的死亡率极高的肝病症候群,严重威胁着人类的健康。据统计显示,我国每年因为重症肝功能衰竭导致死亡的人数为30-50万。治疗肝衰竭最有效的方法是肝移植,然而由于供肝来源缺乏,不到30%的患者能得到肝移植的机会。近年来,人们发现肝干细胞移植对急慢性肝功能衰竭有明确的治疗作用。肝干细胞(Hepatic stem cells,HSC)是体内存在的一种具有自我增殖能力和多向分化潜能的干细胞,能分化为肝细胞和胆管上皮细胞等多种细胞。肝干细胞可参与肝脏的修复与重建,也是肝细胞移植、生物人工肝的重要细胞来源。目前认为,肝干细胞的来源包括:胚胎干细胞、造血干细胞、骨髓间充质干细胞、胚胎肝干细胞、成体肝卵圆细胞及小肝细胞等。尽管许多文献报道不同来源的肝干细胞在体内外均可分化为具有一定功能的肝细胞,但是分化效率报道不一,细胞的终末分化及移植替代功能仍然未得到解决。研究发现,利用基因调控手段上调HSC分化基因表达,可改善其移植治疗效果。胚胎肝干细胞(Embryonic liver stem cells,ELSC)属于肝前体细胞,仅向肝细胞和胆管上皮细胞分化发育,这种中间状态是各种来源的干细胞向成熟肝细胞分化所必经的。胚胎肝干细胞分化机制的研究有助于阐明肝脏的发育机制,促进干细胞分化为成熟肝细胞,防止向肿瘤细胞分化,推进肝干细胞的临床应用。Wnt信号转导途径参与了细胞增殖、分化、生长、迁徙及氧化应激等多方面的复杂信号级联反应,在肝脏发育和肝细胞分化调控中具有重要作用。目前对Wnt信号通路与肝脏发育和肝细胞分化中的研究,主要集中于β-catenin,对Wnt信号其他成员,特别是Wnt拮抗因子的研究甚少。Wnt信号分子在肝脏发育过程中总的表达情况及变化趋势也未见报道。本课题以Wnt信号转导途径与肝脏发育的重要性为理论依据,以胚胎肝干细胞为研究对象进行以下研究:第一部分Wnt信号分子在不同发育阶段肝组织及细胞株中的差异性表达目的:检测不同发育阶段的肝组织及不同分化阶段肝细胞中所有Wnt配体、受体及拮抗因子的表达,分析Wnt信号分子在肝脏发育中的表达差异和变化趋势,为进一步探讨Wnt信号转导途径在肝脏发育及肝细胞分化中的作用提供重要信息。方法:分离胚胎12.5天至出生后4周共9个阶段的小鼠肝脏,建立胚胎肝干细胞株ELSC14-19及肝细胞株LC14d。提取RNA,RT-PCR检测19种Wnt配体,10种Fzd受体,2种共受体及8种Wnt拮抗因子的表达水平,分析变化趋势。结果:在不同阶段的肝组织中能检测到大多数Wnt配体基因的表达,其中Wnt2b、3a、4、8a、9b、10a和10b在几乎所有的肝脏发育阶段中有较高的表达,Wnt2和5a在晚期阶段表达,而Wnt6、16在早期阶段表达。所有的Fzd受体在肝脏组织中被检测到,随着肝脏发育,Fzd2、3、5、6、7、8表达呈现降低趋势,而Fzd1和4在出生后表达升高。LRP5、6在所有的肝脏中也能检测,LRP5持续高表达,LRP6在出生前低表达,出生后增高。SFRP1、4、5在不同阶段的肝组织中的表达无明显差异,SFRP1、4的表达稍高于SFRP5。SFRP2只在E12.5和14.5天的肝组织中表达,随后很快消失。Frzb/SFRP3只在E12.5至18.5天的肝脏中检测到,且表达水平较SFRP2低。DKK2仅在早期肝组织中检测到,而DKK1和DKK3在所有的肝组织中有较低表达,并无明显升高或降低的趋势。在ELSC14-19和LC14d中,Wnt配体的表达基本一致,能检测到9个Wnt基因的表达。Hepa1-6中,Wnt1和Wnt10a高表达,Wnt2消失,Wnt3a和Wnt6出现。大多数的Fzd受体和LRP5、6在ELSC14-19中高表达。5个SFRP拮抗因子中,SFRP2、4高表达,1、5低表达,Frzb在ELSC14-19中表达很低,在Hepa1-6中检测不到。DKK2在ELSC14-19中高表达,DKK1、3表达较低。SFRP2,DKK2在ELSC14-19中的表达高于14d,而SFRP4相反。Wnt信号下游靶基因c-Myc、Axin2、Sox9及Nanog在ELSC14-19中有表达。结论:Wnt信号转导途径在肝脏发育中被激活,Wnt信号各分子呈现差异性表达,其表达趋势在肝组织和细胞中不完全一致,正常肝细胞与肝肿瘤细胞的Wnt信号分子表达差异明显,提示不同的Wnt信号转导通路在肝脏发育和肝细胞分化中可能发挥不同的作用。其中Wnt拮抗因子SFRP2、Frzb、DKK2仅在发育早期的肝组织中检测到,其表达下调可能是肝脏发育和肝细胞分化所必需。第二部分胚胎肝干细胞体外诱导分化的研究目的:构建携带ALB全长启动子及Gaussia荧光素酶报告基因的胚胎肝干细胞,用于肝细胞体外诱导分化的动态检测及诱导方法的筛选。检测不同诱导因素对胚胎肝干细胞体外成熟分化的影响,优化诱导方案。方法:构建携带ALB全长启动子及Gaussia荧光素酶报告基因的逆转录病毒质粒pBGLuc-ALB,转染ELSC14-19、LC14d、Hepa1-6细胞,检测培养上清中的GLuc活性,并与ALB表达趋势比较。pBGLuc-ALB与pAmpho共转染HEK293细胞包装逆转录病毒,感染ELSC14-19构建稳定细胞株。ALB-GLuc活性检测10%FBS、2%HS、Dex、HGF、FGF4单一因素及不同配伍培养条件对胚胎肝干细胞体外成熟分化的影响,RT-PCR、免疫荧光、糖原染色及ICG摄取释放试验检测2%HS+Dex+HGF+FGF4体外联合诱导ELSC14-19成熟分化。结果:Gaussia荧光素酶活性与ALB表达趋势一致。胚胎肝干细胞在2%HS培养中增殖速度低于10%FBS,但是ALB-GLuc的表达更高,HGF、FGF4分别在10ng/ml、20ng/ml浓度下对胚胎肝干细胞ALB-GLuc的诱导作用最强,FGF4能促进细胞增殖。不同因素的配伍组合中,2%HS+0.1uM Dex+10ng/mlHGF+20ng/mlFGF4联合培养能最强诱导ALB表达。诱导后细胞DLK、AFP、CK19表达下调,ALB、CK18、UGT1A表达增强,诱导后第3天可以检测到晚期指标TAT的表达,第6天可以检测到ApoB的表达。糖原合成和ICG摄取释放功能在诱导第6天开始出现,并逐渐增强。结论:构建了携带ALB全长启动子及Gaussia荧光素酶报告基因的胚胎肝干细胞,ALB-GLuc活性能真实地反应细胞中ALB的表达水平。2%HS+0.1uMDex+10ng/mlHGF+20ng/mlFGF4联合培养能有效诱导胚胎肝干细胞体外成熟分化。第三部分Wnt拮抗因子对胚胎肝干细胞分化的影响及机制研究目的:检测并比较Wnt拮抗因子DKK2和Frzb对胚胎肝干细胞体外成熟分化的影响,并探讨其机制。方法:腺病毒DKK2、Frzb分别感染ELSC14-19,并用2%HS+Dex+HGF+FGF4体外诱导培养。于诱导后第12天,RT-PCR和WesternBlot检测肝细胞相关标志,糖原染色及ICG摄取释放试验检测肝细胞功能。pTop-Luc质粒转染ELSC14-19,用β-catenin,19种Wnt配体腺病毒分别配对DKK2,Frzb腺病毒共感染细胞,荧光素酶报告基因检测β-catenin活性。结果:Frzb作用后的ELSC14-19细胞,在体外诱导培养下,DLK、AFP、CK19表达较诱导对照组高,ALB、CK18、UGT1A、TAT、ApoB表达降低,糖原合成及ICG摄取功能阳性细胞比例明显减少,而DKK2处理组与对照组相比无明显差别。β-catenin,Wnt1、2、3、3a、7a、7b、10b处理组GLuc活性明显高于对照组。Frzb作用下β-catenin,Wnt1、2、3、3a、7a、7b诱导的Top-Luc活性降低,而Wnt10b诱导的Luc信号不受影响。DKK2作用下β-catenin,Wnt1、2、3、3a、7b诱导的Top-Luc活性降低,对Wnt 7a诱导的Luc增强没有抑制作用,而对Wnt10b诱导的Luc活性有轻微促进作用。总的来说,Frzb对Wnt配体诱导β-catenin活性的抑制作用强于DKK2。结论:Frzb能够抑制由2%HS+Dex+HGF+FGF4联合诱导的胚胎肝干细胞成熟分化,而DKK2没有影响。其机制可能在于Frzb和DKK2对Wnt信号转导途径的抑制作用不同。更多还原

【Abstract】 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.更多还原