上调肝细胞核因子4α基因表达抑制实验性肝纤维化
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摘要
【研究背景及目的】
肝纤维化(hepatic fibrosis)是肝脏对慢性损伤的一种修复反应,以细胞外基质(extracellular matrix,ECM)在肝内过多沉积为特征。肝纤维化为一动态过程,属可逆性病变,因此,阻断、抑制或逆转肝纤维化是治疗慢性肝病的一个重要目标。
既往认为,肝纤维化发生的中心环节是肝星状细胞(hepatic stellate cell,HSC)激活并向肌成纤维样细胞(myofibroblasts,MFs)转化,抑制HSC激活、增殖与迁移、诱导凋亡是肝纤维化治疗的重要策略。近年来研究发现,在静息状态下,HSC可能是一种上皮细胞,对肝干细胞的分化和增殖以及肝细胞功能维持均起重要作用。此外也有研究显示,肝实质细胞如肝细胞以及胆管上皮细胞均可能转化为MFs,参与肝纤维化的发生发展。因此,抑制HSC活化和增殖可能不是治疗肝纤维化的最好策略,迫切需要针对肝纤维化发病的新机制重新确立新的有效治疗方法。
上皮细胞间质转型(epithelial-to-mesenchymal transition,EMT)主要是指上皮细胞在细胞形态、细胞结构、细胞功能以及细胞粘附、迁移能力等方面获得间质细胞特性的过程。目前,EMT在肝脏、肾脏、肺脏等器官纤维化进程中的重要作用已经得到证实。一系列研究也表明,肝细胞、HSC、胆管上皮细胞也通过EMT转化为MFs,参与肝纤维化进程。这些研究重新认识了肝脏实质细胞和间质细胞作用,是肝纤维化机制与治疗领域新的突破。
肝细胞核因子4(hepatocyte nuclear factor,HNF4)是一种属于细胞核激素受体家族的转录因子,在分化成熟的肝细胞中高表达,其中HNF4α是HNF4的重要亚型。HNF4α参与维护肝细胞脂肪代谢、白蛋白合成、药物解毒、能量代谢、胆汁酸合成等重要功能,也是调控上皮细胞表型(如紧密连接、粘附连接、缝隙连接、桥粒以及细胞与细胞基质间粘附分子、上皮细胞极性和细胞骨架蛋白等)表达的重要基因。
晚近研究发现,TGF-β1可诱导小鼠肝细胞发生EMT并伴有HNF4α表达下调;上调HNF4α表达可阻断EMT重要转录因子Snail基因诱导的肝细胞EMT发生;上调间质细胞NIH-3T3中HNF4α表达可使其向上皮细胞转型(mesenchymal-to-epithelial transition,MET),表明HNF4α对EMT有重要的调控作用。基于以上研究结果我们推测,HNF4α将可能阻断肝纤维化进程中肝细胞通过EMT转化为MFs,从而维持其上皮细胞表型和肝细胞功能。更为重要的是,上调HNF4α基因在HSC中表达,将可能促使活化HSC向上皮细胞表型转化,从而达到既抑制活化HSC的问质细胞特性,又发挥其促进肝脏再生和肝干细胞分化的作用。
本课题在明确肝纤维化进程中HNF4α表达下降的基础上,将携带HNF4α的重组腺病毒—AdHNF4α经尾静脉注射导入肝纤维化大鼠体内,明确上调HNF4α基因表达对实验性肝纤维化的抑制作用;通过上调经TGF-β1刺激后原代培养肝细胞和活化HSC HNF4α的表达,检测上述细胞EMT相关基因和生物学功能变化,阐述HNF4α通过阻断EMT进而抑制肝纤维化的作用机制,为肝纤维化的发病机制和治疗研究提供新的思路。
【实验方法】
一、HNF4α抑制肝纤维化进程中EMT的体内研究
1.利用AdEasy~(TM)系统构建表达HNF4α的重组腺病毒—AdHNF4α,293细胞包装并反复感染,扩增高效表达HNF4α的AdHNF4α和空白对照病毒AdGFP,浓缩纯化、测定滴度后保存。
2.AdHNF4α抑制DMN损伤大鼠的肝纤维化进程
将雄性SD(Sprague-Dawley)大鼠随机分为4组,第1组为正常对照组(n=10);2-4组为肝纤维化模型组,予1%二甲基亚硝胺(dimethylnitrosamine,DMN)以10μg/kg腹腔注射,每周连续注射3次,共注射5w。其中第2组设为模型对照组(n=30),第3组为AdGFP导入组(n=10),第4组设为AdHNF4α导入组(n=10)。分别在第2w、4w处死模型组大鼠各10只。于DMN注射4w后AdHNF4α组和AdGFP组分别经尾静脉导入4×10~9 pfu/只AdHNF4α或4×10~9 pfu/只AdGFP,注射病毒2w后处死大鼠,分别观察各组大鼠肝功能指标及凝血酶原时间(prothrombin time,PT)变化;免疫组织化学法测定肝组织Ⅰ型胶原、Ⅲ型胶原表达;HE和VG染色测定ECM含量并对肝纤维化程度分级,图象分析仪半定量分析。
3.AdHNF4α对胆管结扎(bile duct ligation,BDL)大鼠肝纤维化进程的抑制作用
将雄性SD大鼠随机分为4组,每组12只,分设假手术组、胆总管结扎组、空白病毒AdGFP导入组和AdHNF4α导入组。钝性分离并结扎胆总管,制备BDL肝纤维化模型。术后2d AdGFP组和AdHNF4α组分别经尾静脉导入4×10~9 pfu/只AdGFP或4×10~9 pfu/只AdHNF4α;3w后处死大鼠,观察指标同DMN模型。
4.Real time RT-PCR和免疫组织化学方法,分别检测正常、DMN肝损伤2w和4w大鼠肝组织HNF4α及EMT相关基因的表达;同时检测HNF4α导入后HNF4α及EMT相关基因的表达,评估对实验性肝纤维化的抑制效果。
二、上调HNF4α表达对肝细胞EMT的作用
分离制备原代培养SD大鼠肝细胞,培养2d后,换成含有2ng/ml TGF-β1无血清培养基,同时将AdGFP或AdHNF4α以感染复数(multiplicity of infection,MOI)10感染肝细胞,动态观察细胞形态变化;收集感染48h、72h后细胞,抽提总RNA和蛋白。Real time RT-PCR法检测肝细胞HNF4α、白蛋白(albumin,ALB)、谷胺酰氨合成酶(glutamine synthetase,GS)、细胞色素P450家族1A2(c)rtochrome P4501A2,CYP1A2)、上皮细胞钙粘蛋白(E-cadherin)、波形蛋白(Vimentin)、Snail、Ⅰ型胶原mRNA水平表达。
三、上调HNF4α表达对HSC细胞表型和生物学活性的影响
AdGFP或AdHNF4α分别以MOI 50感染大鼠肝星状细胞株HSC-T6,动态观察细胞形态变化;收集感染后48h、72h细胞,抽提总RNA和蛋白。Real time RT-PCR法检测细胞HNF4α、ALB、GS、CYP1A2、E-cadherin、Vimentin、Snail、Ⅰ型胶原、Ⅲ型胶原、α-SMA、组织金属蛋白酶抑制-1(tissue inhibitor of metalloproteinase-1,TIMP-1)基因mRNA水平表达变化;细胞免疫荧光方法检测HNF4α、Vimentin表达;绘制感染前后细胞增殖曲线。
四、统计学处理
数据采用SPSS 11.0统计软件包进行分析,结果以(?)±S表示。多组间采用One-WayANOVA分析,方差非齐性则采用非参数检验;P＜0.05为具有显著性差异,P＜0.01为具有非常显著性差异。
【实验结果】
一、HNF4α体内导入对实验性肝纤维化的抑制作用
1.DMN损伤肝纤维化进程中EMT相关基因表达变化:Real time RT-PCR检测DMN注射2w和4w大鼠肝组织中HNF4α和上皮细胞表型基因E-cadherin mRNA水平表达较正常组明显降低(P＜0.05),同时肝细胞功能基因白蛋白mRNA水平表达也明显下调(P＜0.01);而间质细胞表型基因Snail、Vimentin表达均较正常组明显增加(P＜0.05)。
2.HNF4α对DMN肝纤维化模型的抑制作用:HNF4α基因导入组血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)及PT值分别为113.2±21.6 U/L、23.8±1.41s,较模型对照组(163.8±19.4 U/L、29.4±1.63 s)和AdGFP组(165.2±27.1 U/L、27.4±1.96s)明显降低(P＜0.05);HNF4α基因导入组肝组织羟脯氨酸含量为217.16±42.51μg/g,较模型对照组(341.07±81.20μg/g)和AdGFP组(361.37±112.83μg/g)明显减少(P＜0.05)。HNF4α组胶原染色面积约为AdGFP组的49%(P＜0.05),免疫组化表明Ⅰ型胶原、Ⅲ型胶原表达较AdGFP组均显著下降(P＜0.05)。
3.HNF4α对BDL模型肝纤维化的抑制作用:HNF4α基因导入组血清ALT、AST分别为86.4±13.9U/L、523.6±90.0 U/L,较AdGFP组(127.8±13.9 U/L、726.8±129.7 U/L)有明显差异(P＜0.05);治疗组胶原染色面积约为AdGFP组的52%(P＜0.05),免疫组化表明Ⅰ型胶原、Ⅲ型胶原表达较AdGFP组均显著下降(P＜0.05)。
4.HNF4α对DMN损伤肝纤维化模型肝组织ALB和EMT相关基因表达影响:AdHNF4α导入组ALB和E-cadherin mRNA表达较AdGFP导入组及模型组显著增加(P＜0.01),而Snail、Vimentin表达均显著减少(P＜0.05)。
二、HNF4α对TGFβ1诱导原代培养肝细胞EMT的影响
1.将含有TGF-β1无血清培养基刺激原代大鼠肝细胞24 h后,肝细胞形态无明显变化;48 h后可见较多死亡细胞,细胞数量减少,形态由多边形向梭形转变;72 h后见死亡细胞进一步增多,活细胞数量较少且大多数呈现间质细胞梭形形态。Realtime RT-PCR检测Vimentin、Snail、Ⅰ型胶原表达均明显增强(P＜0.05),而E-cadherin、HNF4α、ALB、GS、CYP1A2表达明显减少(P＜0.05)。
2.将MOI 10的AdHNF4α和AdGFP分别感染TGF-β1刺激的原代大鼠肝细胞,24 h后,两组在细胞数量和细胞形态上无明显差别,荧光显微镜下均可见大量GFP表达;48 h后AdHNF4α组细胞死亡数量较AdGFP组明显减少,细胞形态仍保持多边形:72 h后AdGFP组活细胞数量较少且大多数呈现间质细胞梭形形态,AdHNF4α组细胞死亡细胞很少且大多仍保持多边形形态。Real time RT-PCR检测AdHNF4α组HNF4α表达明显上调,Vimentin、Snail和Ⅰ型胶原表达均明显减少(P＜0.05),E-cadherin明显增加(P＜0.05),肝细胞功能基因ALB、GS、CYP1A2表达明显增强(P＜0.05)。
三、HNF4α对HSC细胞表型和生物学活性的影响
1.将AdHNF4α以MOI 50滴度感染肝星状细胞株HSC-T6 48 h和72 h后AdHNF4α组Vimentin、Snail、Ⅰ型胶原、α-SMA和TIMP-1 mRNA水平均明显下降(P＜0.05),E-cadherin表达明显增加(P＜0.05),GS和CYP1A2等肝细胞功能基因明显增强(P＜0.05);72h后AFP mRNA及HNF4α蛋白表达明显增加,Vimentin蛋白表达减弱。
2.MTS法连续5d测定OD_(450)值,发现HNF4α导入HSC-T6后,细胞增殖明显抑制,第4d OD_(450)值为0.9761±0.1151,增殖活性显著低于对照组(阴性对照组1.4427±0.1567,AdGFP组1.3403±0.1120,P＜0.01)。
【结论】
1.肝纤维化进程中,肝细胞HNF4α表达逐渐降低。
2.AdHNF4α体内基因导入可明显抑制大鼠肝脏ECM沉积,促进肝功能恢复,是治疗实验性肝纤维化新的有效方法。
3.HNF4α基因导入大鼠肝细胞后可阻断TGF-β1诱导的EMT,维持肝细胞正常表型,增强肝细胞功能。
4.HNF4α基因导入可使活化HSC向上皮细胞转型,并抑制其MFs生物学功能。
5.HNF4α治疗肝纤维化的主要机制是抑制肝细胞和HSC的EMT,恢复纤维化肝脏中肝细胞功能,并抑制活化HSC生物学活性。
【Background and Objective】
Hepatic fibrosis,characterized by deposition of extracellular matrix(ECM) in the Disse's space,is a pathological response to a variety of chronic liver diseases,which is recognized as a dynamic and reversal process.Therefore,blocking,inhibition or even reversal of hepatic fibrosis is a major target for the treatment of chronic liver disease.
Generally,it has been accepted that activated hepatic stellate cells(HSC), accompanying phenotypic transformation into myofibroblast-like cells,play the pivotal role in hepatic fibrogenesis.In the past decades,it is an important target on hepatic fibrosis therapy by repressing activation,proliferation and migration of HSC as well as inducing its apoptosis.Recently,several studies reported that HSC could represent a epithelial phenotype and play a significant role in inducing liver stem cell differentiation, maintaining hepatocyte proliferation and function.In addition,it has been reported that hepatic parenchymal cells,such as hepatocytes and biliary epithelial cells,participated in the process of hepatic fibrosis.Therefore,it is unreasonable to repress the activation and proliferation of HSC in hepatic fibrosis therapy,and new strategy on additional fibrotic mechanism and treatment should be reestablished.
Epithelial-to-mesenchymal transition(EMT) is the process that epithelial cells gradually lose their epithelial signatures while acquiring the characteristics of mesenchymal cells in cell morphology,structure,biological function,adhesion and migration ability.More importantly,some researches revealed that EMT was a predominant mediator to participate in the process of organ fibrosis in kidney,lung and liver.Substantial advances have been made to highlight that during fibrosis,the heterogeneous pool of(myo-)fibroblasts can be supplemented by EMT from cholangiocytes and potentially also from hepatocytes.Therefore,we believed that extension of EMT concepts in the hepatic fibrogenesis will provide a new therapeutic target for hepatic fibrosis.
Hepatocyte nuclear factor 4(HNF4) is a member of the nuclear hormone receptor family of transcription factors.HNF4αis a main phenotype of HNF4 which plays an important role not only in regulating hepatocyte differentiation and maintaining liver-specific functions including energy metabolism,xenobiotic detoxification,bile acid synthesis,serum protein production,but also in controlling epithelial phenotype of hepatocyte.Importantly,HNF4αregulates the expression of genes encoding proteins involved in all major types of cell junctions including tight junctions,adhesion junctions, gap junctions,desmosomes,as well as epithelial polarization and cytoskeletal organization.
Recently,some studies demonstrated that expression of HNF4αwas closely associated with EMT.The expression of HNF4αdecreased obviously while transforming growth factor-β1(TGF-β1) induced an EMT state in mouse hepatocytes in vitro. Overexpression of HNF4αcould inhibit the process of EMT induced by Snail.In addition, its ectopic expression in fibroblasts induces a mesenchymal-to-epithelial transition, suggesting that HNF4αwas a dominant regulator of the epithelial phenotype.Based on the above results,we presume that HNF4αmight interrupt EMT of hepatocytes to maintain its epithelial phenotype and biological function;more importantly,its ectopic expression may induce MET of the activated HSC as well as facilitate hepatocytes regeneration.
In this study,we detected the expression of HNF4αin the process of hepatic fibrosis and examine the effect of HNF4αon hepatic fibrosis by tail vein injection of AdHNF4α. Furthermore,we detected expression of EMT related genes in rat hepatocytes stimulated by TGF-β1 as well as activated HSC after AdHNF4αinfection in vitro,in order to elucidate the anti-fibrotic mechanism of HNF4α,which would provide a noval effective strategy for the treatment of hepatic fibrosis.
【Methods】
1.HNF4αeffect on experimental hepatic fibrosis
AdHNF4αand control adenovirus-AdGFP replication-deficient recombinant adenoviruses were packaged in 293 cells respectively.Then adenoviruses were stored after purification by cesium chloride(CsCl) gradient centrifugation and determination of the viral titers.
Dimethylnitrosamine(DMN) induced model:Sixty male Sprague-Dawley(SD) rats were divided into 4 groups randomly.Group 1(n=10) was served as a normal control,and the rats in next three groups(n=50) were hepatic fibrosis models induced by 1%DMN(10μg/kg) three times per week for 5 weeks intraperitoneally.Ten rats were sacrificed at 2 and 4 weeks respectively after DMN injection.Four weeks after DMN injection,rats in group 2 (10 rats),3(n=10) and 4(n=10) were infused with saline,4×10~9 pfu AdGFP or 4×10~9 pfu AdHNF4αvia tail vein,respectively.At the end of study(2 weeks after gene delivery),the animals were sacrificed for analysis of liver function and histology.Immunohistochemical staining was used to detect collagen typeⅠandⅢ.For the semiquantitative analysis, stained sections were measured by an image analyzer.
Bile duct ligation(BDL) induced model:The 48 male SD rats were divided into 4 groups randomly.Each group composed 12 rats.Group 1 was treated with sham surgery served as the control,and the rats in next three groups were hepatic fibrosis models induced by BDL for 3 weeks.Common bile ducts of models were dissected bluntly and ligated.After ligated for 2 days,rats in group 2,3,4 were infused with saline,4×10~9 pfu AdGFP or 4×10~9 pfu AdHNF4αvia tail vein injection,respectively.Three weeks after BDL,the animals were sacrificed for examinations of liver function and immunochemistry.
Expression of HNF4αin the normal liver and the fibrotic liver induced by DMN was detected by real time RT-PCR and immunohistochemistry staining.In addition,mRNA expression of EMT related genes was detected by real time RT-PCR.
2.Effects of HNF4αgene on hepatocytes
Primary hepatocytes derived from male SD rats were cultured for 2 days,then the culture medium was replaced by serum-free medium containing 2 ng/ml TGFβ1. Simulately,the adenoviruses AdGFP or AdHNF4αwere added with 10 MOI.Cell morphology was observed at different time points.Total RNA and protein in different groups were extracted and the expression of HNF4α,albumin(ALB),glutamine synthetase (GS),cytochrome P4501A2(CYP1A2),E-cadherin,Vimentin,Snail and collagenⅠmRNA in the cells was detected by real time RT-PCR respectively after adenovirus infection for 48 and 72 hours.
3.Effects of HNF4αon HSC cell phenotype and biological characteristics
The rat HSC cell line HSC-T6 was infected by AdGFP and AdHNF4αwith 50 MOI respectively.Cell morphology was observed at different time points.Total RNA and protein in different groups were extracted and the expression of HNF4α,ALB,GS, CYP1A2,E-cadherin,Vimentin,HNF4α,Snail,collagen typeⅠ,α-SMA and tissue inhibitor of metalloproteinase-1(TIMP-1) mRNA in the cells was detected by real time RT-PCR after adenovirus infection for 48 and 72 hours.On the other hand,cell proliferation in HNF4αtransfection group was compared with that in the control using MTS method.
4.Statistical Analysis
Statistical analysis of values was performed with SPSS software(11.0 version),with a P value＜0.05 considered significant and P＜0.01 as very significant.
【Results】
1.HNF4αeffect on experimental hepatic fibrosis
(1) Real time RT-PCR and immunohistochemistry staining indicated that the expression of HNF4αin the fibrotic livers decreased gradually after DMN injection, compared with that of the normal liver.In addition,the expression of Vimentin and Snail was enhanced in the fibrotic liver,while expression of E-cadherin decreased significantly (P＜0.05).
(2) HNF4αinhibits the development of hepatic fibrosis induced by DMN
Serum level of ALT in AdHNF4αinfusion group,saline infusion group and AdGFP infusion group was 113.2±21.6 U/L,163.8±19.4 U/L and 165.2±27.1 U/L,respectively (P＜0.05).In addition,PT level reduced in AdHNF4αgroup(23.8±1.41 s),compared with that in saline group(29.4±1.63 s) and AdGFP group(27.4±1.96 s).Furthermore,less amount of liver hydroxyproline was detected in AdHNF4αgroup(217.16±42.51μg/g), compared with that of saline infusion group(341.07±81.20μg/g) and AdGFP group (361.37±112.83μg/g)(P＜0.05).Based on the analysis of image scan technique,we found that the area of ECM in AdHNF4αgroup reduced to 49%of that in AdGFP group.The expression of collagen typeⅠand typeⅢdecreased significantly after AdHNF4αinfusion.
(3) HNF4αsuppresses hepatic fibrosis induced by BDL
In AdHNF4αinfusion group,serum levels of ALT and AST(86.4±13.9 U/L, 523.6±90.0 U/L) decreased significantly compared with that in AdGFP infusion group (127.8±13.9 U/L,726.8±129.7 U/L,P＜0.05).Semiquantitative analysis revealed that AdHNF4αtreatment reduced fibrotic areas by 48%(P＜0.05).Moreover,the expression of collagen typeⅠandⅢalso decreased significantly after AdHNF4αinfusion.
(4) HNF4αinhibits the process of EMT in liver tissue
Real time-RT PCR demonstrated that the expression of Vimentin and Snail in liver infected with AdHNF4α,was down-regulated compared with that in AdGFP treated liver, while E-cadherin expression was up-regulated significantly(P＜0.05).
2.Effects of HNF4αon hepatocytes EMT
(1) Hepatocytes morphology had no obvious changes after TGF-β1 addition for 24 hours.TGF-β1-treated hepatocytes represented a spindle-shaped morphology 48 hours later.After TGF-β1 stimulation for 72 hours,the alteration of hepatocytes morphology became more obviously.Real time-PCR indicated that the expression of Vimentin,Snail and collgen typeⅠmRNA was up-regulated in TGF-β1-treated hepatocyte(P＜0.05),while the expression of E-cadherin,HNF4α,ALB,GS and CYP1A2 was suppressed(P＜0.05). These results demonstrated that TGF-β1 could induce an EMT state in rat primary hepatocyte in vitro.
(2) Cellular morphology of hepatocytes treated by TGF-β1+AdHNF4αpreserved a more cuboidal/hexagonal shape compared with that of TGF-β1+AdGFP treated ones.In addition,the number of viable hepatocytes in TGF-β1+AdGFP group reduced significantly. Real time-PCR showed that the expression of mesenchymal phenotype genes in AdHNF4αinfected hepatocytes,including Vimentin,Snail and collgen typeⅠmRNA,was down-regulated(P＜0.05).On the other hand,the expression of liver-specific genes and epithelial phenotype genes such as HNF4α,ALB,GS,CYP1A2 and E-cadherin were up-regulated significantly(P＜0.05).These results imply that HNF4αcould interrupt the EMT process of primary hepatocyte induced by TGF-β1.
3.Effects of HNF4αon HSC cell phenotype and biological characteristics
Real time-PCR revealed that the expression of Vimentin,Snail,collagenⅠ,α-SMA and TIMP-1 decreased significantly in HSC-T6 infected with AdHNF4α(P＜0.05). Interestingly,the expression of liver-specific genes including GS,CYP1A2 and AFP enhanced(P＜0.05).More importantly,E-cadherin expression(epithelial phenotype gene) was also elevated(P＜0.05) after AdHNF4αinfection.Cellular morphology of activated HSC treated by AdHNF4αcould turn from a spindle shape into a more cuboidal/hexagonal shape.Additionally,proliferation of HSC-T6 by HNF4αgene delivery was inhibited according to MTS method(P＜0.01).
【Conclusion】
All of our results revealed that:
1.The expression of HNF4αwas gradually down-regulated in the development of hepatic fibrosis.
2.Gene delivery of HNF4αin vivo was an effective strategy to attenuate experimental hepatic fibrosis by reducing ECM deposition and promoting hepatocyte function.
3.HNF4αcould interrupt EMT process in TGF-β1 treated hepatocytes,preserve their epithelial morphology,as well as improve hepatocyte function.
4.HNF4αcould inhibit the biological characteristic of HSC and up-regulate liver-specific genes by inducing MET process of the activated HSC,.
5.HNF4αameliorates hepatic fibrosis by interrupting EMT of hepatocytes and HSCs, which could lead to improve hepatocyte function and inhibit the activity of activated HSCs.
肝纤维化(hepatic fibrosis)是肝脏对慢性损伤的一种修复反应,以细胞外基质(extracellular matrix,ECM)在肝内过多沉积为特征。肝纤维化为一动态过程,属可逆性病变,因此,阻断、抑制或逆转肝纤维化是治疗慢性肝病的一个重要目标。
既往认为,肝纤维化发生的中心环节是肝星状细胞(hepatic stellate cell,HSC)激活并向肌成纤维样细胞(myofibroblasts,MFs)转化,抑制HSC激活、增殖与迁移、诱导凋亡是肝纤维化治疗的重要策略。近年来研究发现,在静息状态下,HSC可能是一种上皮细胞,对肝干细胞的分化和增殖以及肝细胞功能维持均起重要作用。此外也有研究显示,肝实质细胞如肝细胞以及胆管上皮细胞均可能转化为MFs,参与肝纤维化的发生发展。因此,抑制HSC活化和增殖可能不是治疗肝纤维化的最好策略,迫切需要针对肝纤维化发病的新机制重新确立新的有效治疗方法。
上皮细胞间质转型(epithelial-to-mesenchymal transition,EMT)主要是指上皮细胞在细胞形态、细胞结构、细胞功能以及细胞粘附、迁移能力等方面获得间质细胞特性的过程。目前,EMT在肝脏、肾脏、肺脏等器官纤维化进程中的重要作用已经得到证实。一系列研究也表明,肝细胞、HSC、胆管上皮细胞也通过EMT转化为MFs,参与肝纤维化进程。这些研究重新认识了肝脏实质细胞和间质细胞作用,是肝纤维化机制与治疗领域新的突破。
肝细胞核因子4(hepatocyte nuclear factor,HNF4)是一种属于细胞核激素受体家族的转录因子,在分化成熟的肝细胞中高表达,其中HNF4α是HNF4的重要亚型。HNF4α参与维护肝细胞脂肪代谢、白蛋白合成、药物解毒、能量代谢、胆汁酸合成等重要功能,也是调控上皮细胞表型(如紧密连接、粘附连接、缝隙连接、桥粒以及细胞与细胞基质间粘附分子、上皮细胞极性和细胞骨架蛋白等)表达的重要基因。
晚近研究发现,TGF-β1可诱导小鼠肝细胞发生EMT并伴有HNF4α表达下调;上调HNF4α表达可阻断EMT重要转录因子Snail基因诱导的肝细胞EMT发生;上调间质细胞NIH-3T3中HNF4α表达可使其向上皮细胞转型(mesenchymal-to-epithelial transition,MET),表明HNF4α对EMT有重要的调控作用。基于以上研究结果我们推测,HNF4α将可能阻断肝纤维化进程中肝细胞通过EMT转化为MFs,从而维持其上皮细胞表型和肝细胞功能。更为重要的是,上调HNF4α基因在HSC中表达,将可能促使活化HSC向上皮细胞表型转化,从而达到既抑制活化HSC的问质细胞特性,又发挥其促进肝脏再生和肝干细胞分化的作用。
本课题在明确肝纤维化进程中HNF4α表达下降的基础上,将携带HNF4α的重组腺病毒—AdHNF4α经尾静脉注射导入肝纤维化大鼠体内,明确上调HNF4α基因表达对实验性肝纤维化的抑制作用;通过上调经TGF-β1刺激后原代培养肝细胞和活化HSC HNF4α的表达,检测上述细胞EMT相关基因和生物学功能变化,阐述HNF4α通过阻断EMT进而抑制肝纤维化的作用机制,为肝纤维化的发病机制和治疗研究提供新的思路。
【实验方法】
一、HNF4α抑制肝纤维化进程中EMT的体内研究
1.利用AdEasy~(TM)系统构建表达HNF4α的重组腺病毒—AdHNF4α,293细胞包装并反复感染,扩增高效表达HNF4α的AdHNF4α和空白对照病毒AdGFP,浓缩纯化、测定滴度后保存。
2.AdHNF4α抑制DMN损伤大鼠的肝纤维化进程
将雄性SD(Sprague-Dawley)大鼠随机分为4组,第1组为正常对照组(n=10);2-4组为肝纤维化模型组,予1%二甲基亚硝胺(dimethylnitrosamine,DMN)以10μg/kg腹腔注射,每周连续注射3次,共注射5w。其中第2组设为模型对照组(n=30),第3组为AdGFP导入组(n=10),第4组设为AdHNF4α导入组(n=10)。分别在第2w、4w处死模型组大鼠各10只。于DMN注射4w后AdHNF4α组和AdGFP组分别经尾静脉导入4×10~9 pfu/只AdHNF4α或4×10~9 pfu/只AdGFP,注射病毒2w后处死大鼠,分别观察各组大鼠肝功能指标及凝血酶原时间(prothrombin time,PT)变化;免疫组织化学法测定肝组织Ⅰ型胶原、Ⅲ型胶原表达;HE和VG染色测定ECM含量并对肝纤维化程度分级,图象分析仪半定量分析。
3.AdHNF4α对胆管结扎(bile duct ligation,BDL)大鼠肝纤维化进程的抑制作用
将雄性SD大鼠随机分为4组,每组12只,分设假手术组、胆总管结扎组、空白病毒AdGFP导入组和AdHNF4α导入组。钝性分离并结扎胆总管,制备BDL肝纤维化模型。术后2d AdGFP组和AdHNF4α组分别经尾静脉导入4×10~9 pfu/只AdGFP或4×10~9 pfu/只AdHNF4α;3w后处死大鼠,观察指标同DMN模型。
4.Real time RT-PCR和免疫组织化学方法,分别检测正常、DMN肝损伤2w和4w大鼠肝组织HNF4α及EMT相关基因的表达;同时检测HNF4α导入后HNF4α及EMT相关基因的表达,评估对实验性肝纤维化的抑制效果。
二、上调HNF4α表达对肝细胞EMT的作用
分离制备原代培养SD大鼠肝细胞,培养2d后,换成含有2ng/ml TGF-β1无血清培养基,同时将AdGFP或AdHNF4α以感染复数(multiplicity of infection,MOI)10感染肝细胞,动态观察细胞形态变化;收集感染48h、72h后细胞,抽提总RNA和蛋白。Real time RT-PCR法检测肝细胞HNF4α、白蛋白(albumin,ALB)、谷胺酰氨合成酶(glutamine synthetase,GS)、细胞色素P450家族1A2(c)rtochrome P4501A2,CYP1A2)、上皮细胞钙粘蛋白(E-cadherin)、波形蛋白(Vimentin)、Snail、Ⅰ型胶原mRNA水平表达。
三、上调HNF4α表达对HSC细胞表型和生物学活性的影响
AdGFP或AdHNF4α分别以MOI 50感染大鼠肝星状细胞株HSC-T6,动态观察细胞形态变化;收集感染后48h、72h细胞,抽提总RNA和蛋白。Real time RT-PCR法检测细胞HNF4α、ALB、GS、CYP1A2、E-cadherin、Vimentin、Snail、Ⅰ型胶原、Ⅲ型胶原、α-SMA、组织金属蛋白酶抑制-1(tissue inhibitor of metalloproteinase-1,TIMP-1)基因mRNA水平表达变化;细胞免疫荧光方法检测HNF4α、Vimentin表达;绘制感染前后细胞增殖曲线。
四、统计学处理
数据采用SPSS 11.0统计软件包进行分析,结果以(?)±S表示。多组间采用One-WayANOVA分析,方差非齐性则采用非参数检验;P＜0.05为具有显著性差异,P＜0.01为具有非常显著性差异。
【实验结果】
一、HNF4α体内导入对实验性肝纤维化的抑制作用
1.DMN损伤肝纤维化进程中EMT相关基因表达变化:Real time RT-PCR检测DMN注射2w和4w大鼠肝组织中HNF4α和上皮细胞表型基因E-cadherin mRNA水平表达较正常组明显降低(P＜0.05),同时肝细胞功能基因白蛋白mRNA水平表达也明显下调(P＜0.01);而间质细胞表型基因Snail、Vimentin表达均较正常组明显增加(P＜0.05)。
2.HNF4α对DMN肝纤维化模型的抑制作用:HNF4α基因导入组血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)及PT值分别为113.2±21.6 U/L、23.8±1.41s,较模型对照组(163.8±19.4 U/L、29.4±1.63 s)和AdGFP组(165.2±27.1 U/L、27.4±1.96s)明显降低(P＜0.05);HNF4α基因导入组肝组织羟脯氨酸含量为217.16±42.51μg/g,较模型对照组(341.07±81.20μg/g)和AdGFP组(361.37±112.83μg/g)明显减少(P＜0.05)。HNF4α组胶原染色面积约为AdGFP组的49%(P＜0.05),免疫组化表明Ⅰ型胶原、Ⅲ型胶原表达较AdGFP组均显著下降(P＜0.05)。
3.HNF4α对BDL模型肝纤维化的抑制作用:HNF4α基因导入组血清ALT、AST分别为86.4±13.9U/L、523.6±90.0 U/L,较AdGFP组(127.8±13.9 U/L、726.8±129.7 U/L)有明显差异(P＜0.05);治疗组胶原染色面积约为AdGFP组的52%(P＜0.05),免疫组化表明Ⅰ型胶原、Ⅲ型胶原表达较AdGFP组均显著下降(P＜0.05)。
4.HNF4α对DMN损伤肝纤维化模型肝组织ALB和EMT相关基因表达影响:AdHNF4α导入组ALB和E-cadherin mRNA表达较AdGFP导入组及模型组显著增加(P＜0.01),而Snail、Vimentin表达均显著减少(P＜0.05)。
二、HNF4α对TGFβ1诱导原代培养肝细胞EMT的影响
1.将含有TGF-β1无血清培养基刺激原代大鼠肝细胞24 h后,肝细胞形态无明显变化;48 h后可见较多死亡细胞,细胞数量减少,形态由多边形向梭形转变;72 h后见死亡细胞进一步增多,活细胞数量较少且大多数呈现间质细胞梭形形态。Realtime RT-PCR检测Vimentin、Snail、Ⅰ型胶原表达均明显增强(P＜0.05),而E-cadherin、HNF4α、ALB、GS、CYP1A2表达明显减少(P＜0.05)。
2.将MOI 10的AdHNF4α和AdGFP分别感染TGF-β1刺激的原代大鼠肝细胞,24 h后,两组在细胞数量和细胞形态上无明显差别,荧光显微镜下均可见大量GFP表达;48 h后AdHNF4α组细胞死亡数量较AdGFP组明显减少,细胞形态仍保持多边形:72 h后AdGFP组活细胞数量较少且大多数呈现间质细胞梭形形态,AdHNF4α组细胞死亡细胞很少且大多仍保持多边形形态。Real time RT-PCR检测AdHNF4α组HNF4α表达明显上调,Vimentin、Snail和Ⅰ型胶原表达均明显减少(P＜0.05),E-cadherin明显增加(P＜0.05),肝细胞功能基因ALB、GS、CYP1A2表达明显增强(P＜0.05)。
三、HNF4α对HSC细胞表型和生物学活性的影响
1.将AdHNF4α以MOI 50滴度感染肝星状细胞株HSC-T6 48 h和72 h后AdHNF4α组Vimentin、Snail、Ⅰ型胶原、α-SMA和TIMP-1 mRNA水平均明显下降(P＜0.05),E-cadherin表达明显增加(P＜0.05),GS和CYP1A2等肝细胞功能基因明显增强(P＜0.05);72h后AFP mRNA及HNF4α蛋白表达明显增加,Vimentin蛋白表达减弱。
2.MTS法连续5d测定OD_(450)值,发现HNF4α导入HSC-T6后,细胞增殖明显抑制,第4d OD_(450)值为0.9761±0.1151,增殖活性显著低于对照组(阴性对照组1.4427±0.1567,AdGFP组1.3403±0.1120,P＜0.01)。
【结论】
1.肝纤维化进程中,肝细胞HNF4α表达逐渐降低。
2.AdHNF4α体内基因导入可明显抑制大鼠肝脏ECM沉积,促进肝功能恢复,是治疗实验性肝纤维化新的有效方法。
3.HNF4α基因导入大鼠肝细胞后可阻断TGF-β1诱导的EMT,维持肝细胞正常表型,增强肝细胞功能。
4.HNF4α基因导入可使活化HSC向上皮细胞转型,并抑制其MFs生物学功能。
5.HNF4α治疗肝纤维化的主要机制是抑制肝细胞和HSC的EMT,恢复纤维化肝脏中肝细胞功能,并抑制活化HSC生物学活性。
【Background and Objective】
Hepatic fibrosis,characterized by deposition of extracellular matrix(ECM) in the Disse's space,is a pathological response to a variety of chronic liver diseases,which is recognized as a dynamic and reversal process.Therefore,blocking,inhibition or even reversal of hepatic fibrosis is a major target for the treatment of chronic liver disease.
Generally,it has been accepted that activated hepatic stellate cells(HSC), accompanying phenotypic transformation into myofibroblast-like cells,play the pivotal role in hepatic fibrogenesis.In the past decades,it is an important target on hepatic fibrosis therapy by repressing activation,proliferation and migration of HSC as well as inducing its apoptosis.Recently,several studies reported that HSC could represent a epithelial phenotype and play a significant role in inducing liver stem cell differentiation, maintaining hepatocyte proliferation and function.In addition,it has been reported that hepatic parenchymal cells,such as hepatocytes and biliary epithelial cells,participated in the process of hepatic fibrosis.Therefore,it is unreasonable to repress the activation and proliferation of HSC in hepatic fibrosis therapy,and new strategy on additional fibrotic mechanism and treatment should be reestablished.
Epithelial-to-mesenchymal transition(EMT) is the process that epithelial cells gradually lose their epithelial signatures while acquiring the characteristics of mesenchymal cells in cell morphology,structure,biological function,adhesion and migration ability.More importantly,some researches revealed that EMT was a predominant mediator to participate in the process of organ fibrosis in kidney,lung and liver.Substantial advances have been made to highlight that during fibrosis,the heterogeneous pool of(myo-)fibroblasts can be supplemented by EMT from cholangiocytes and potentially also from hepatocytes.Therefore,we believed that extension of EMT concepts in the hepatic fibrogenesis will provide a new therapeutic target for hepatic fibrosis.
Hepatocyte nuclear factor 4(HNF4) is a member of the nuclear hormone receptor family of transcription factors.HNF4αis a main phenotype of HNF4 which plays an important role not only in regulating hepatocyte differentiation and maintaining liver-specific functions including energy metabolism,xenobiotic detoxification,bile acid synthesis,serum protein production,but also in controlling epithelial phenotype of hepatocyte.Importantly,HNF4αregulates the expression of genes encoding proteins involved in all major types of cell junctions including tight junctions,adhesion junctions, gap junctions,desmosomes,as well as epithelial polarization and cytoskeletal organization.
Recently,some studies demonstrated that expression of HNF4αwas closely associated with EMT.The expression of HNF4αdecreased obviously while transforming growth factor-β1(TGF-β1) induced an EMT state in mouse hepatocytes in vitro. Overexpression of HNF4αcould inhibit the process of EMT induced by Snail.In addition, its ectopic expression in fibroblasts induces a mesenchymal-to-epithelial transition, suggesting that HNF4αwas a dominant regulator of the epithelial phenotype.Based on the above results,we presume that HNF4αmight interrupt EMT of hepatocytes to maintain its epithelial phenotype and biological function;more importantly,its ectopic expression may induce MET of the activated HSC as well as facilitate hepatocytes regeneration.
In this study,we detected the expression of HNF4αin the process of hepatic fibrosis and examine the effect of HNF4αon hepatic fibrosis by tail vein injection of AdHNF4α. Furthermore,we detected expression of EMT related genes in rat hepatocytes stimulated by TGF-β1 as well as activated HSC after AdHNF4αinfection in vitro,in order to elucidate the anti-fibrotic mechanism of HNF4α,which would provide a noval effective strategy for the treatment of hepatic fibrosis.
【Methods】
1.HNF4αeffect on experimental hepatic fibrosis
AdHNF4αand control adenovirus-AdGFP replication-deficient recombinant adenoviruses were packaged in 293 cells respectively.Then adenoviruses were stored after purification by cesium chloride(CsCl) gradient centrifugation and determination of the viral titers.
Dimethylnitrosamine(DMN) induced model:Sixty male Sprague-Dawley(SD) rats were divided into 4 groups randomly.Group 1(n=10) was served as a normal control,and the rats in next three groups(n=50) were hepatic fibrosis models induced by 1%DMN(10μg/kg) three times per week for 5 weeks intraperitoneally.Ten rats were sacrificed at 2 and 4 weeks respectively after DMN injection.Four weeks after DMN injection,rats in group 2 (10 rats),3(n=10) and 4(n=10) were infused with saline,4×10~9 pfu AdGFP or 4×10~9 pfu AdHNF4αvia tail vein,respectively.At the end of study(2 weeks after gene delivery),the animals were sacrificed for analysis of liver function and histology.Immunohistochemical staining was used to detect collagen typeⅠandⅢ.For the semiquantitative analysis, stained sections were measured by an image analyzer.
Bile duct ligation(BDL) induced model:The 48 male SD rats were divided into 4 groups randomly.Each group composed 12 rats.Group 1 was treated with sham surgery served as the control,and the rats in next three groups were hepatic fibrosis models induced by BDL for 3 weeks.Common bile ducts of models were dissected bluntly and ligated.After ligated for 2 days,rats in group 2,3,4 were infused with saline,4×10~9 pfu AdGFP or 4×10~9 pfu AdHNF4αvia tail vein injection,respectively.Three weeks after BDL,the animals were sacrificed for examinations of liver function and immunochemistry.
Expression of HNF4αin the normal liver and the fibrotic liver induced by DMN was detected by real time RT-PCR and immunohistochemistry staining.In addition,mRNA expression of EMT related genes was detected by real time RT-PCR.
2.Effects of HNF4αgene on hepatocytes
Primary hepatocytes derived from male SD rats were cultured for 2 days,then the culture medium was replaced by serum-free medium containing 2 ng/ml TGFβ1. Simulately,the adenoviruses AdGFP or AdHNF4αwere added with 10 MOI.Cell morphology was observed at different time points.Total RNA and protein in different groups were extracted and the expression of HNF4α,albumin(ALB),glutamine synthetase (GS),cytochrome P4501A2(CYP1A2),E-cadherin,Vimentin,Snail and collagenⅠmRNA in the cells was detected by real time RT-PCR respectively after adenovirus infection for 48 and 72 hours.
3.Effects of HNF4αon HSC cell phenotype and biological characteristics
The rat HSC cell line HSC-T6 was infected by AdGFP and AdHNF4αwith 50 MOI respectively.Cell morphology was observed at different time points.Total RNA and protein in different groups were extracted and the expression of HNF4α,ALB,GS, CYP1A2,E-cadherin,Vimentin,HNF4α,Snail,collagen typeⅠ,α-SMA and tissue inhibitor of metalloproteinase-1(TIMP-1) mRNA in the cells was detected by real time RT-PCR after adenovirus infection for 48 and 72 hours.On the other hand,cell proliferation in HNF4αtransfection group was compared with that in the control using MTS method.
4.Statistical Analysis
Statistical analysis of values was performed with SPSS software(11.0 version),with a P value＜0.05 considered significant and P＜0.01 as very significant.
【Results】
1.HNF4αeffect on experimental hepatic fibrosis
(1) Real time RT-PCR and immunohistochemistry staining indicated that the expression of HNF4αin the fibrotic livers decreased gradually after DMN injection, compared with that of the normal liver.In addition,the expression of Vimentin and Snail was enhanced in the fibrotic liver,while expression of E-cadherin decreased significantly (P＜0.05).
(2) HNF4αinhibits the development of hepatic fibrosis induced by DMN
Serum level of ALT in AdHNF4αinfusion group,saline infusion group and AdGFP infusion group was 113.2±21.6 U/L,163.8±19.4 U/L and 165.2±27.1 U/L,respectively (P＜0.05).In addition,PT level reduced in AdHNF4αgroup(23.8±1.41 s),compared with that in saline group(29.4±1.63 s) and AdGFP group(27.4±1.96 s).Furthermore,less amount of liver hydroxyproline was detected in AdHNF4αgroup(217.16±42.51μg/g), compared with that of saline infusion group(341.07±81.20μg/g) and AdGFP group (361.37±112.83μg/g)(P＜0.05).Based on the analysis of image scan technique,we found that the area of ECM in AdHNF4αgroup reduced to 49%of that in AdGFP group.The expression of collagen typeⅠand typeⅢdecreased significantly after AdHNF4αinfusion.
(3) HNF4αsuppresses hepatic fibrosis induced by BDL
In AdHNF4αinfusion group,serum levels of ALT and AST(86.4±13.9 U/L, 523.6±90.0 U/L) decreased significantly compared with that in AdGFP infusion group (127.8±13.9 U/L,726.8±129.7 U/L,P＜0.05).Semiquantitative analysis revealed that AdHNF4αtreatment reduced fibrotic areas by 48%(P＜0.05).Moreover,the expression of collagen typeⅠandⅢalso decreased significantly after AdHNF4αinfusion.
(4) HNF4αinhibits the process of EMT in liver tissue
Real time-RT PCR demonstrated that the expression of Vimentin and Snail in liver infected with AdHNF4α,was down-regulated compared with that in AdGFP treated liver, while E-cadherin expression was up-regulated significantly(P＜0.05).
2.Effects of HNF4αon hepatocytes EMT
(1) Hepatocytes morphology had no obvious changes after TGF-β1 addition for 24 hours.TGF-β1-treated hepatocytes represented a spindle-shaped morphology 48 hours later.After TGF-β1 stimulation for 72 hours,the alteration of hepatocytes morphology became more obviously.Real time-PCR indicated that the expression of Vimentin,Snail and collgen typeⅠmRNA was up-regulated in TGF-β1-treated hepatocyte(P＜0.05),while the expression of E-cadherin,HNF4α,ALB,GS and CYP1A2 was suppressed(P＜0.05). These results demonstrated that TGF-β1 could induce an EMT state in rat primary hepatocyte in vitro.
(2) Cellular morphology of hepatocytes treated by TGF-β1+AdHNF4αpreserved a more cuboidal/hexagonal shape compared with that of TGF-β1+AdGFP treated ones.In addition,the number of viable hepatocytes in TGF-β1+AdGFP group reduced significantly. Real time-PCR showed that the expression of mesenchymal phenotype genes in AdHNF4αinfected hepatocytes,including Vimentin,Snail and collgen typeⅠmRNA,was down-regulated(P＜0.05).On the other hand,the expression of liver-specific genes and epithelial phenotype genes such as HNF4α,ALB,GS,CYP1A2 and E-cadherin were up-regulated significantly(P＜0.05).These results imply that HNF4αcould interrupt the EMT process of primary hepatocyte induced by TGF-β1.
3.Effects of HNF4αon HSC cell phenotype and biological characteristics
Real time-PCR revealed that the expression of Vimentin,Snail,collagenⅠ,α-SMA and TIMP-1 decreased significantly in HSC-T6 infected with AdHNF4α(P＜0.05). Interestingly,the expression of liver-specific genes including GS,CYP1A2 and AFP enhanced(P＜0.05).More importantly,E-cadherin expression(epithelial phenotype gene) was also elevated(P＜0.05) after AdHNF4αinfection.Cellular morphology of activated HSC treated by AdHNF4αcould turn from a spindle shape into a more cuboidal/hexagonal shape.Additionally,proliferation of HSC-T6 by HNF4αgene delivery was inhibited according to MTS method(P＜0.01).
【Conclusion】
All of our results revealed that:
1.The expression of HNF4αwas gradually down-regulated in the development of hepatic fibrosis.
2.Gene delivery of HNF4αin vivo was an effective strategy to attenuate experimental hepatic fibrosis by reducing ECM deposition and promoting hepatocyte function.
3.HNF4αcould interrupt EMT process in TGF-β1 treated hepatocytes,preserve their epithelial morphology,as well as improve hepatocyte function.
4.HNF4αcould inhibit the biological characteristic of HSC and up-regulate liver-specific genes by inducing MET process of the activated HSC,.
5.HNF4αameliorates hepatic fibrosis by interrupting EMT of hepatocytes and HSCs, which could lead to improve hepatocyte function and inhibit the activity of activated HSCs.
引文
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2.Lee JM,Dedhar S,Kalluri R,et al.The epithelial-rnesenchymal transition:new insights in signaling,development,and disease.J Cell Biol,2006,172(7);973-981.
3.Thiery JP,Sleeman JP.Complex networks orchestrate epithelial-mesenchymal transitions.Nat Rev Mol Cell Biol,2006,7(2):131-142.
4.Zeisberg M,Yang C,Martino M,et al.Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition.J Biol Chem,2007,282(32):23337-23347.
5.Zeisberg M,Hanai J,Sugimoto H,et al.BMP-7 counteracts TGF-betal-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med, 2003, 9(7): 964-968.
6. Willis BC, duBois RM, Borok Z. Epithelial origin of myofibroblasts during fibrosis in the lung. Proc Am Thorac Soc, 2006, 3(4): 377-382.
7. Pagan R, Sanchez A, Martin I, et al. Effects of growth and differentiation factors on the epithelial-mesenchymal transition in cultured neonatal rat hepatocytes. J Hepatol, 1999, 31(5): 895-904.
8. Valdes F, Alvarez AM, Locascio A, et al. The epithelial mesenchymal transition confers resistance to the apoptotic effects of transforming growth factor Beta in fetal rat hepatocytes. Mol Cancer Res, 2002,1(1): 68-78.
9. Cicchini C, Laudadio I, Citarella F, et al. TGFp-induced EMT requires focal adhesion kinase (FAK) signaling. Exp Cell Res, 2008,314(1): 143-152.
10. Kaimori A, Potter J, Kaimori JY, et al. Transforming Growth Factor-beta1 Induces an Epithelial-to-Mesenchymal Transition State in Mouse Hepatocytes in Vitro. J Biol Chem, 2007,282(30): 22089-220101.
11. Kojima T, Takano K, Yamamoto T, et al. Transforming growth factor-P induces epithelial to mesenchymal transition by down-regulation of claudin-1 expression and the fence function in adult rat hepatocytes. Liver Int, 2008,28(4): 534-545.
12. Lim YS, Kim KA, Jung JO, et al. Modulation of cytokeratin expression during in vitro cultivation of human hepatic stellate cells: evidence of transdifferentiation from epithelial to mesenchymal phenotype. Histochem Cell Biol, 2002,118:127-136.
13. Lim, YS, HC Lee, HS Lee. Switch of cadherin expression from E- to N-type during the activation of rat hepatic stellate cells. Histochem Cell Biol, 2007,127(2): 149-160.
14. Sicklick JK, Choi SS, Bustamante M, et al. Evidence for epithelial-mesenchymal transitions in adult liver cells. Am J Physiol Gastrointest Liver Physiol, 2006, 291(4): 575-583.
15. Kordes C, Sawitza I, Muller-Marbach A, et al. CD133+ hepatic stellate cells are progenitor cells. Biochem Biophys Res Commun. 2007, 352(2): 410-417.
16. Xia JL, Dai C, Michalopoulos GK, et al. Hepatocyte growth factor attenuates liver fibrosis induced by bile duct ligation. Am J Pathol, 2006,168(5): 1500-1512.
17. Rygiel KA, Robertson H, Marshall HL, et al. Epithelial-mesenchymal transition contributes to portal tract fibrogenesis during human chronic liver disease. Lab Invest, 2008, 88(2): 112-123.
18. Sato Y, Harada K, Ozaki S, et al. Cholangiocytes with mesenchymal features contribute to progressive hepatic fibrosis of the polycystic kidney rat. Am J Pathol, 2007, 171(6): 1859-1871.
19. Robertson H, Kirby JA, Yip WW, et al. Biliary epithelial-mesenchymal transition in posttransplantation recurrence of primary biliary cirrhosis. Hepatology, 2007, 45(4): 977-981.
20. Diaz R, Kim JW, Hui JJ, et al. Evidence for the epithelial to mesenchymal transition in biliary atresia fibrosis. Hum Pathol, 2008, 39(1): 102-115.
21. Kisseleva T, Brenner DA. Mechanisms of Fibrogenesis. Exp Biol Med, 2008, 233(2): 109-122.
22. Ju W, Ogawa A, Heyer J, et al. Deletion of Smad2 in mouse liver reveals novel functions in hepatocyte growth and differentiation. Mol Cell Biol, 2006, 26(2): 654-667.
23. Watermann DO, Gabriel B, Jager M, et al. Specific induction of pp125 focal adhesion kinase in human breast cancer. Br J Cancer, 2005, 93(6):694-698.
24. McLean GW, Carragher NO, Avizienyte E, et al. The role of focal-adhesion kinase in cancer - a new therapeutic opportunity. Nat Rev Cancer, 2005, 5(7): 505-515.
25. Zavadil J, Bottinger EP. TGF-β and epithelial-to-mesenchymal transitions. Oncogene, 2005, 24(37): 5764-5774.
26. Ohkubo T, Ozawa M. The transcription factor Snail downregulates the tight junction components independently of E-cadherin downregulation. J Cell Scil, 2004, 117(Pt 9): 1675-1685.
27. Bolos V, Peinado H, Perez-Moreno MA, et al. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J Cell Sci. 2003,116(Pt 3): 499-511.
28. Zavadil J, Cermak L, Soto-Nieves N, et al. Integration of TGF-beta/Smad and Jagged1/Notch signalling in epithelial-to-mesenchymal transition. EMBO J, 2004 , 23(5): 1155-1165.
29. Cicchini C, Filippini D, Coen S, et al. Snail controls differentiation of hepatocytes by repressing HNF4alpha expression. J Cell Physiol, 2006, 209(1): 230-238.
30. Gressner OA, Weiskirchen R, Gressner AM. Evolving concepts of liver fibrogenesis provide new diagnostic and therapeutic options. Comp Hepatol, 2007, 6(1): 7.
31. Weiskirchen R, Meurer SK. Bone morphogenetic protein-7 in focus: a member of the transforming growth factor-beta superfamily is implicated in the maintenance of liver health. Hepatology, 2007,45(5): 1324-1325.
32. Kowanetz M, Valcourt U, Bergstrom R, et al. Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor beta and bone morphogenetic protein. Mol Cell Biol. 2004,24(10): 4241-4254.
33. Kinoshita K, Iimuro Y, Otogawa K, Adenovirus-mediated expression of BMP-7 suppresses the development of liver fibrosis in rats. Gut, 2007, 56(5) :706-714.
1. Iredale JP. Cirrhosis: new research provides a basis for rational and targeted treatments. BMJ. 2003, 327(7407): 143-147.
2. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest, 2005,115(2): 209-218.
3. Friedman SL. The cellular basis of hepatic fibrosis. Mechanism and treatment strategies. N Engl J Med, 1993, 328: 1828-1835.
4. Lim, YS, HC Lee, HS Lee. Switch of cadherin expression from E- to N-type during the activation of rat hepatic stellate cells. Histochem Cell Biol, 2007,127(2): 149-160.
5. Sicklick JK, Choi SS, Bustamante M, et al. Evidence for epithelial-mesenchymal transitions in adult liver cells. Am J Physiol Gastrointest Liver Physiol, 2006, 291(4): 575-583.
6. Friedman SL. Hepatic Stellate Cells: Protean, Multifunctional, and Enigmatic Cells of the Liver. Physiol Rev, 2008, 88(1):125-172.
7. Baba S, Fujii H, Hirose T, et al. Commitment of bone marrow cells to hepatic stellate cells in mouse. J Hepatol, 2004,40: 255-260.
8. Antoine M, Wirz W, Tag CG, et al. Expression and function of fibroblast growth factor (FGF) 9 in hepatic stellate cells and its role in toxic liver injury. Biochem Biophys Res Commun, 2007, 361: 335-341.
9. Kubota H, Yao H, Reid LM. Identification and characterization of vitamin-A storing cells in fetal liver: implication of functional importance of hepatic stellate cells in development and hematopoiesis. Stem Cells, 2007,25: 2339-2349.
10. Zeisberg M, Yang C, Martino M, et al. Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition. J Biol Chem, 2007, 282(32): 23337-23347.
11. Xia JL, Dai C, Michalopoulos GK, et al. Hepatocyte growth factor attenuates liver fibrosis induced by bile duct ligation. Am J Pathol, 2006, 168(5): 1500-1512.
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