肝脏祖细胞/卵圆细胞在肝硬化及肝癌中的增殖、与肝脏微环境关系及部分生物学特性研究
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摘要
第一部分人肝硬化及肝癌中肝脏祖细胞的激活与扩增一肝硬化中肝脏祖细胞的激活与扩增
目的证实肝硬化组织中存在人肝脏祖细胞(HPCs),探讨HPCs分布及激活的强度与肝脏炎症程度的关系,提供HPCs向肝细胞分化的依据。
方法对30例肝硬化及3例正常组织标本进行常规组织学观察,对门脉炎症程度进行评分,并用胆管上皮标志CK7和肝星状细胞激活标志SMA进行免疫组化染色,对符合HPCs、中间型肝细胞以及小管样反应的细胞进行计数和半定量评分。
结果在正常肝组织中无门脉周围HPCs和小管样反应增殖。在肝硬化组织中,增殖的HPCs起源于肝门脉区域,随着门脉炎症程度加重,HPCs及小管样反应从肝硬化结节周围向肝实质扩散并出现中间型肝细胞增殖,其周围有显著的肝星状细胞激活。HPCs及小管样反应增殖程度随着门脉炎症程度的加重而增加。HPCs数目与中间型肝细胞数目之间存在正直线相关。ALT和AST与HPCs及中间型肝细胞数目存在正直线相关。
结论在人肝硬化中存在祖细胞的激活,炎症反应是HPCs激活的触发因素,HPCs向肝实质内迁移并向肝细胞方向分化是肝再生的重要途径。
二肝硬化中肝脏祖细胞的表型特征研究
目的研究不同肝脏祖细胞标志物在人肝硬化组织中的定位和分布,明确肝脏祖细胞群的免疫表型特征。
方法用OV-6,CK7,CK19,Hepatocyte, c-kit以及AFP对30例肝硬化及3例正常肝组织进行免疫组化染色和激光共聚焦免疫荧光双标,观察这些标志物在不同细胞群中的分布。
结果正常肝脏中,胆管和胆小管上皮CK7及CK19阳性,OV-6及c-kit阴性。在肝硬化中,门脉周围区域的肝脏祖细胞(HPCs)及小管样反应细胞主要为OV-6(+)/ CK19(+)/CK7(+)/ Hepatocyte(+),少数为OV-6(-)。随着门脉及实质内炎症的加重,HPCs及小管样反应从肝硬化结节周围向肝实质扩散并出现中间型肝细胞增殖。HPCs及小管样反应细胞主要为OV-6(-)/CK19(+)/CK7(+),Hepatocyte(-)或(+);中间型肝细胞为OV-6(-)/CK19(-)/CK7(+)/ Hepatocyte(+),少数Hepatocyte(-)。门脉周围区域和纤维间隔内存在少量的c-kit(+)细胞,个别阳性细胞整合到成熟胆管,其余c-kit(+)细胞不共表达OV-6、CK7及CK19。
结论人肝硬化存在肝脏祖细胞的激活;肝脏祖细胞群的免疫表型差异表明祖细胞处于不同的分化增殖阶段;不同肝脏祖细胞标志物的联合运用是鉴别和研究肝脏祖细胞的有力工具。
三c-kit、CK7在人肝细胞肝癌中的表达及意义
目的研究肝脏祖细胞标志物c-kit、CK7在肝细胞肝癌(HCC)组织中的表达及其与临床病理的关系。
方法对40例肝细胞肝癌标本及3例正常组织标本进行常规组织学观察以及c-kit、CK7、CD45免疫组化染色,对肿瘤细胞的分化程度进行分型,分析c-kit、CK7表达与肝细胞肝癌的临床病理特征的联系。
结果正常肝脏中c-kit染色阴性。19/40例HCC组织中存在c-kit(+)肿瘤细胞,在肿瘤细胞之间或肿瘤结节周围分散分布。30例(75%)存在CK7阳性癌细胞,阳性程度强弱不一,主要存在两种不同免疫反应形态特征。HBsAg及Anti-HBc在c-kit(+)及CK7(+)HCC表达更常见(p<0.05),c-kit、CK7表达与肿瘤的分化程度紧密相关(p<0.05)。
结论骨髓来源的肝脏祖细胞参与了HCC的形成和发展,c-kit、CK7对于判断HCC预后有一定意义。
第二部分肝脏微环境参与卵圆细胞介导的肝再生
目的阐明在卵圆细胞介导的肝再生中肝脏非实质细胞、胞外基质成分与卵圆细胞的相互作用,分析肝脏微环境对卵圆细胞的调控作用。
方法采用免疫组化及免疫荧光双标的方法动态观察在2-AAF/PH大鼠卵圆细胞增殖模型中,随着卵圆细胞的增殖和分化,Kupffer细胞、肝星状细胞、肝脏胞外基质成分(层粘连蛋白和纤维连接蛋白)的定位及与卵圆细胞的相互关系。
结果PH后第2天,小卵圆细胞开始向门静脉周围区域增殖。Desmin(+)的HSCs及laminin主要出现在门静脉周围的肝窦状隙内,而Kupffer细胞和fibronectin在整个肝小叶内表达显著增加。从术后第4到第9天,卵圆细胞进一步向肝实质内增殖,与HSCs、laminin及fibronectin关系密切,Kupffer细胞与卵圆细胞混合在一起增殖,第6天以后开始减少。从术后第12到第15天,随着卵圆细胞分化为小肝细胞节结,大多数HSCs、laminin及fibronectin位于节结周边,少数出现在节结内,Kupffer细胞主要出现在中央静脉周围区域。第18天以后,正常的肝小叶结构开始恢复。
结论在肝损伤后的重建修复过程中,卵圆细胞与肝脏非实质细胞、胞外基质成分存在紧密的联系,局部肝脏微环境可能通过细胞与细胞之间以及细胞与基质之间的相互作用在卵圆细胞介导的肝再生中发挥重要的调控作用。
第三部分大鼠肝卵圆细胞的分离、鉴定、培养及端粒酶的表达
目的建立大鼠卵圆细胞增殖模型,探讨其分离、鉴定、培养的方法;检测卵圆细胞端粒酶活性及相关基因表达,探讨与其增殖分化之间的关系。
方法采用2-AAF/PH模型诱导大鼠卵圆细胞增殖,2-AAF剂量为15mg/kg,于术后9-12天采用改良的胶原酶灌注消化密度梯度离心法分离卵圆细胞,用电镜、RT-PCR、细胞免疫荧光鉴定卵圆细胞,采用免疫组化、RT-PCR及端粒酶活性检测试剂盒检测分离的卵圆细胞及卵圆细胞系LE6端粒酶的表达,并分析其意义。
结果采用2-AAF/PH模型成功诱导大鼠卵圆细胞增殖。分离的卵圆细胞核大,卵圆形,胞浆少,呈铺路石样生长,细胞直径约7～12μm,电镜观察发现有少量绒毛样突起,核浆比例大,胞浆内细胞器少且发育不成熟。细胞免疫荧光、RT-PCR结果显示表达0V-6、AFP、CK19、Albumin、C-KIT、CK18、Thy-1。免疫组织化学显示端粒酶催化亚单位TERT表达在门静脉周围增殖的卵圆细胞细胞核内,随着卵圆细胞逐渐向肝细胞方向分化,TERT阳性细胞数目显著较少。分离的卵圆细胞及卵圆细胞系均表达端粒酶亚单位TERT及TR,与LE6相比,刚分离的卵圆细胞表达水平更低;随着LE6传代次数的增加,端粒酶活性逐渐降低。
结论采用剂量为15mg/kg的2-AAF/PH模型可诱导卵圆细胞大量增殖;改良的胶原酶灌注消化、密度梯度离心的分离方法可获取纯度较高的卵圆细胞;端粒酶活性可能是卵圆细胞维持其增殖能力和多分化潜能的一个必要条件。
Part I-1 Hepatic Progenitor cell activation and expansion in human liver cirrhosis.
Objective To confirm whether human hepatic progenitor cells(HPCs) occur in human liver cirrhosis,to investigate the relationship between the degree of HPCs activation and the degree of liver inflammation and provide the proof that HPCs differentiate into hepatocyte.
Methods Surgical specimens from 30 cases of human liver cirrhosis and 3 cases of normal liver were investigated by light microscopy and immunohistochemical staining for CK7(a marker of biliary differentiation) and SMA(a marker of hepatic stellate cell activation).The degree of portal inflammation were determines on routine stained sections.The number of HPCs and intermediate hepatocyte and the extent of the ductular reaction were assessed.
Results HPCs and ductular reaction were not observed in normal livers.In liver cirrhosis the HPCs originate from the portal area.with the increase of the portal inflammation,HPCs and ductular reaction extend from the periphery of liver cirrhosis nodules to the liver parenchyma and the intermediate hepatocyte proliferation were obverved.The notable hepatic stellate cell activation occur around the HPCs and ductular reaction.The number of HPCs and the exent of ductular reaction increased significantly as the portal inflammation increased. There were significant correlations between the number of HPCs with the number of intermediate hepatocyte.In addition,there was strong correlation between the ALT and AST with the number of HPCs and intermediate hepatocyte.
Conclusion Human hepatic progenitor cell activation exist in human liver cirrhosis.The inflammation is a trigger for HPCs activation.HPCs migration from portal area to liver parenchyma and differentiation into hepatocyte are important pathway for liver regeneration.
Part I-2 Phenotype characteristic of human hepatic progenitor cells in human liver cirrhosis.
Objective To identify the phenotype characteristic of human hepatic progenitor cells through investigating the localization and distribution of hepatic progenitor cells markers in human liver cirrhosis.
Methods Surgical specimens from 30 cases of human liver cirrhosis and 3 cases of normal liver were investigated by immunohistochemical staining and double immunofluorescent staining with confocal microscopy for OV-6,CK7,CK19,Hepatocyte,c-kit and AFP.
Results In normal liver,bile duct and ductules were immunostained with CK7 and CK19,whereas OV-6 and c-kit were negative. The HPCs and ductular reaction in the periportal region were OV-6(+)/CK19(+)/CK7(+)/ Hepatocyte(+).Occasionally,they were OV-6(-).With the increase of the portal and liver parenchyma inflammation,HPCs and ductular reaction extended from the periphery of liver cirrhosis nodules to the liver parenchyma and the intermediate hepatocyte proliferation were obverved.The HPCs and ductular reaction were OV-6(-)/CK19(+)/ CK7 (+), Hepatocyte(-)or(+).The intermediate hepatocytes were OV-6(-)/CK19(-)/CK7(+)/Hepatocyte(+),Occasionally,they were Hepatocyte(-).C-kit(+) cells were located in the periportal region and fibrous septa.Very few c-kit-positive cells were found integrated into bile duct,the others didn’t coexpress OV-6,CK7 and CK19.
Conclusion Human hepatic progenitor cells activation existed in human liver cirrhosis;The phenotype difference of different cell populations indicated that hepatic progenitor cells were at different stages of the proliferation and differentiation. Combination of different HPCs markers were powerful tools to identify and investigate the HPCs.
Part I-3 Expression of c-kit and CK7 in human hepatocellular carcinoma
Objective To investigate the expression of hepatic progenitor cell marker c-kit and CK7, to explore the relationship between the expression and the clinicopathologic features in human hepatocellular carcinoma.
Methods Surgical specimens from 40 cases of human hepatocellular carcinoma and 3 cases of normal liver were investigated by HE and immunohistochemical staining for c-kit,CK7 and CD45.The stages of tumor cell differentiations were assessed.
Results In normal liver c-kit was negative. We found c-kit(+) tumor cells scattered individually among the tumor cells or located in the periphery of tumor nodules in 19 of 40 HCC. Immunostaining for CK7 was detected in 30 of 40 HCC,and the positive extent varied from each other. Two different immunostaining features were observed,There were significant differences in the expression of HBsAg and Anti-HBc between the c-kit(+) HCC and c-kit(-) HCC or CK7(+) and CK7(-) HCC (p<0.05).The c-kit and CK7 expression was closely related to the degree of tumor cell differentiation(p<0.05).
Conclusion Bone marrow derived hepatic progenitor cells were involved in the liver regeneration during the course of the development of HCC.C-kit and CK7 expression may be a prognostic indicator for HCC.
PartⅡParticipation of the hepatic non-parenchymal cells and extracellular matrix in oval cell-mediated liver regeneration
Objective The aim of the study was to elucidate the the interaction between non-parenchmal cells,extracellular matrix components and oval cells during the restitutive process.
Methods we examined the localization of oval cell,Kupffer cells,hepatic stellate cells ,and the extracellular matrix components laminin and fibronectin using the immunohistochemistry and double Immunofluorescent analyis during the proliferation and differentiation of oval cells in 2-AAF/PH rats model.
Results By day 2 after PH small oval cells began to proliferate around the portal area.Most of desmin(+) HSCs and laminin were present along the hepatic sinusoids in the periportal area.Kupffer cells and fibronectin markedly increased in the whole hepatic lobule.From day 4 to 9,oval cells spread further into hepatic parenchyma,closely associated with HSCs,fibronectin and laminin. Kupffer cells admixed with oval cell by day 6 and then decreased in the periportal zone. From day 12 to 15 most of HSCs,laminin and fibronectin located around the nodus as the differentiation of oval cells into small hepatocte nodus,and minority of them were present in the nodus.Kupffer cells were mainly liminted in the pericentral sinusoids.After day 18 the normal liver lobules structures began to recover.
Conclusion There is a close relationship between the non-parenchymal cells,ECM components and oval cells during the restitutive repair. Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.
PartⅢIsolation,identification and cultivation of rat oval cells and expression of telomerase in oval cells
Objective To establish the proliferative model of rat oval cells,explore the methods of isolating,identifying and culturing the oval cells;To detect the telomerase activity and the expression of correlated genes in oval cells,explore the relationship with the proliferation and differentiation of oval cells.
Methods 2-AAF/PH rat model was used to induce the proliferation of oval cells.The dose of 2-AAF was 15mg/kg. Oval cells were isolated by the modified collagenase perfusion, digestion and gradient centrifugation. Electron microscope examination,RT-PCR and Immunofluorescence were adopted to identify oval cells. Immunohistochemistry,RT-PCR and telomerase activity detection kit were used to examin the expression of the isolated oval cells and oval cell line LE6.
Results We have successfully induced the proliferation of oval cells through the 2-AAF/PH rat model. Freshly isolated cells showed a oval nuclei,a small proportion of cytoplasm and a cobblestone appearance.The diameter of oval cells was about 7 to 12μm.Electron microscope examination showed that there were villose protuberances,a high nucleus/ cytoplasm ratio,immature organelles.Immunofluorescence staining and RT-PCR showed that oval cells expressed 0V-6、AFP、CK19、Albumin、C-KIT、CK18、Thy-1. Immunohistochemistry showed that TERT was located in the nuclei of oval cells around the portal areas.As oval cells differentiated into small hepatocytes,the number of the TERT(+) cells decreased significantly.Both of the isolated oval cells and oval cell lines LE6 expressed the TERT and telomerase RNA(TR).Compared with LE6,the expression of TERT and TR in the isolated oval cells were much lower.The telomerase activity decreased gradually as the increase of the oval cells passage.
Conclusion The 15mg/kg 2-AAF/PH model can induce a great quantity of oval cells to proliferate.High purity oval cells were available through the modified collagenase perfusion,digestion and gradient centrifugation. Telomerase activity may be indispensable for maintaining the proliferative and multi-directional differentiation abilities of oval cells.
目的证实肝硬化组织中存在人肝脏祖细胞(HPCs),探讨HPCs分布及激活的强度与肝脏炎症程度的关系,提供HPCs向肝细胞分化的依据。
方法对30例肝硬化及3例正常组织标本进行常规组织学观察,对门脉炎症程度进行评分,并用胆管上皮标志CK7和肝星状细胞激活标志SMA进行免疫组化染色,对符合HPCs、中间型肝细胞以及小管样反应的细胞进行计数和半定量评分。
结果在正常肝组织中无门脉周围HPCs和小管样反应增殖。在肝硬化组织中,增殖的HPCs起源于肝门脉区域,随着门脉炎症程度加重,HPCs及小管样反应从肝硬化结节周围向肝实质扩散并出现中间型肝细胞增殖,其周围有显著的肝星状细胞激活。HPCs及小管样反应增殖程度随着门脉炎症程度的加重而增加。HPCs数目与中间型肝细胞数目之间存在正直线相关。ALT和AST与HPCs及中间型肝细胞数目存在正直线相关。
结论在人肝硬化中存在祖细胞的激活,炎症反应是HPCs激活的触发因素,HPCs向肝实质内迁移并向肝细胞方向分化是肝再生的重要途径。
二肝硬化中肝脏祖细胞的表型特征研究
目的研究不同肝脏祖细胞标志物在人肝硬化组织中的定位和分布,明确肝脏祖细胞群的免疫表型特征。
方法用OV-6,CK7,CK19,Hepatocyte, c-kit以及AFP对30例肝硬化及3例正常肝组织进行免疫组化染色和激光共聚焦免疫荧光双标,观察这些标志物在不同细胞群中的分布。
结果正常肝脏中,胆管和胆小管上皮CK7及CK19阳性,OV-6及c-kit阴性。在肝硬化中,门脉周围区域的肝脏祖细胞(HPCs)及小管样反应细胞主要为OV-6(+)/ CK19(+)/CK7(+)/ Hepatocyte(+),少数为OV-6(-)。随着门脉及实质内炎症的加重,HPCs及小管样反应从肝硬化结节周围向肝实质扩散并出现中间型肝细胞增殖。HPCs及小管样反应细胞主要为OV-6(-)/CK19(+)/CK7(+),Hepatocyte(-)或(+);中间型肝细胞为OV-6(-)/CK19(-)/CK7(+)/ Hepatocyte(+),少数Hepatocyte(-)。门脉周围区域和纤维间隔内存在少量的c-kit(+)细胞,个别阳性细胞整合到成熟胆管,其余c-kit(+)细胞不共表达OV-6、CK7及CK19。
结论人肝硬化存在肝脏祖细胞的激活;肝脏祖细胞群的免疫表型差异表明祖细胞处于不同的分化增殖阶段;不同肝脏祖细胞标志物的联合运用是鉴别和研究肝脏祖细胞的有力工具。
三c-kit、CK7在人肝细胞肝癌中的表达及意义
目的研究肝脏祖细胞标志物c-kit、CK7在肝细胞肝癌(HCC)组织中的表达及其与临床病理的关系。
方法对40例肝细胞肝癌标本及3例正常组织标本进行常规组织学观察以及c-kit、CK7、CD45免疫组化染色,对肿瘤细胞的分化程度进行分型,分析c-kit、CK7表达与肝细胞肝癌的临床病理特征的联系。
结果正常肝脏中c-kit染色阴性。19/40例HCC组织中存在c-kit(+)肿瘤细胞,在肿瘤细胞之间或肿瘤结节周围分散分布。30例(75%)存在CK7阳性癌细胞,阳性程度强弱不一,主要存在两种不同免疫反应形态特征。HBsAg及Anti-HBc在c-kit(+)及CK7(+)HCC表达更常见(p<0.05),c-kit、CK7表达与肿瘤的分化程度紧密相关(p<0.05)。
结论骨髓来源的肝脏祖细胞参与了HCC的形成和发展,c-kit、CK7对于判断HCC预后有一定意义。
第二部分肝脏微环境参与卵圆细胞介导的肝再生
目的阐明在卵圆细胞介导的肝再生中肝脏非实质细胞、胞外基质成分与卵圆细胞的相互作用,分析肝脏微环境对卵圆细胞的调控作用。
方法采用免疫组化及免疫荧光双标的方法动态观察在2-AAF/PH大鼠卵圆细胞增殖模型中,随着卵圆细胞的增殖和分化,Kupffer细胞、肝星状细胞、肝脏胞外基质成分(层粘连蛋白和纤维连接蛋白)的定位及与卵圆细胞的相互关系。
结果PH后第2天,小卵圆细胞开始向门静脉周围区域增殖。Desmin(+)的HSCs及laminin主要出现在门静脉周围的肝窦状隙内,而Kupffer细胞和fibronectin在整个肝小叶内表达显著增加。从术后第4到第9天,卵圆细胞进一步向肝实质内增殖,与HSCs、laminin及fibronectin关系密切,Kupffer细胞与卵圆细胞混合在一起增殖,第6天以后开始减少。从术后第12到第15天,随着卵圆细胞分化为小肝细胞节结,大多数HSCs、laminin及fibronectin位于节结周边,少数出现在节结内,Kupffer细胞主要出现在中央静脉周围区域。第18天以后,正常的肝小叶结构开始恢复。
结论在肝损伤后的重建修复过程中,卵圆细胞与肝脏非实质细胞、胞外基质成分存在紧密的联系,局部肝脏微环境可能通过细胞与细胞之间以及细胞与基质之间的相互作用在卵圆细胞介导的肝再生中发挥重要的调控作用。
第三部分大鼠肝卵圆细胞的分离、鉴定、培养及端粒酶的表达
目的建立大鼠卵圆细胞增殖模型,探讨其分离、鉴定、培养的方法;检测卵圆细胞端粒酶活性及相关基因表达,探讨与其增殖分化之间的关系。
方法采用2-AAF/PH模型诱导大鼠卵圆细胞增殖,2-AAF剂量为15mg/kg,于术后9-12天采用改良的胶原酶灌注消化密度梯度离心法分离卵圆细胞,用电镜、RT-PCR、细胞免疫荧光鉴定卵圆细胞,采用免疫组化、RT-PCR及端粒酶活性检测试剂盒检测分离的卵圆细胞及卵圆细胞系LE6端粒酶的表达,并分析其意义。
结果采用2-AAF/PH模型成功诱导大鼠卵圆细胞增殖。分离的卵圆细胞核大,卵圆形,胞浆少,呈铺路石样生长,细胞直径约7～12μm,电镜观察发现有少量绒毛样突起,核浆比例大,胞浆内细胞器少且发育不成熟。细胞免疫荧光、RT-PCR结果显示表达0V-6、AFP、CK19、Albumin、C-KIT、CK18、Thy-1。免疫组织化学显示端粒酶催化亚单位TERT表达在门静脉周围增殖的卵圆细胞细胞核内,随着卵圆细胞逐渐向肝细胞方向分化,TERT阳性细胞数目显著较少。分离的卵圆细胞及卵圆细胞系均表达端粒酶亚单位TERT及TR,与LE6相比,刚分离的卵圆细胞表达水平更低;随着LE6传代次数的增加,端粒酶活性逐渐降低。
结论采用剂量为15mg/kg的2-AAF/PH模型可诱导卵圆细胞大量增殖;改良的胶原酶灌注消化、密度梯度离心的分离方法可获取纯度较高的卵圆细胞;端粒酶活性可能是卵圆细胞维持其增殖能力和多分化潜能的一个必要条件。
Part I-1 Hepatic Progenitor cell activation and expansion in human liver cirrhosis.
Objective To confirm whether human hepatic progenitor cells(HPCs) occur in human liver cirrhosis,to investigate the relationship between the degree of HPCs activation and the degree of liver inflammation and provide the proof that HPCs differentiate into hepatocyte.
Methods Surgical specimens from 30 cases of human liver cirrhosis and 3 cases of normal liver were investigated by light microscopy and immunohistochemical staining for CK7(a marker of biliary differentiation) and SMA(a marker of hepatic stellate cell activation).The degree of portal inflammation were determines on routine stained sections.The number of HPCs and intermediate hepatocyte and the extent of the ductular reaction were assessed.
Results HPCs and ductular reaction were not observed in normal livers.In liver cirrhosis the HPCs originate from the portal area.with the increase of the portal inflammation,HPCs and ductular reaction extend from the periphery of liver cirrhosis nodules to the liver parenchyma and the intermediate hepatocyte proliferation were obverved.The notable hepatic stellate cell activation occur around the HPCs and ductular reaction.The number of HPCs and the exent of ductular reaction increased significantly as the portal inflammation increased. There were significant correlations between the number of HPCs with the number of intermediate hepatocyte.In addition,there was strong correlation between the ALT and AST with the number of HPCs and intermediate hepatocyte.
Conclusion Human hepatic progenitor cell activation exist in human liver cirrhosis.The inflammation is a trigger for HPCs activation.HPCs migration from portal area to liver parenchyma and differentiation into hepatocyte are important pathway for liver regeneration.
Part I-2 Phenotype characteristic of human hepatic progenitor cells in human liver cirrhosis.
Objective To identify the phenotype characteristic of human hepatic progenitor cells through investigating the localization and distribution of hepatic progenitor cells markers in human liver cirrhosis.
Methods Surgical specimens from 30 cases of human liver cirrhosis and 3 cases of normal liver were investigated by immunohistochemical staining and double immunofluorescent staining with confocal microscopy for OV-6,CK7,CK19,Hepatocyte,c-kit and AFP.
Results In normal liver,bile duct and ductules were immunostained with CK7 and CK19,whereas OV-6 and c-kit were negative. The HPCs and ductular reaction in the periportal region were OV-6(+)/CK19(+)/CK7(+)/ Hepatocyte(+).Occasionally,they were OV-6(-).With the increase of the portal and liver parenchyma inflammation,HPCs and ductular reaction extended from the periphery of liver cirrhosis nodules to the liver parenchyma and the intermediate hepatocyte proliferation were obverved.The HPCs and ductular reaction were OV-6(-)/CK19(+)/ CK7 (+), Hepatocyte(-)or(+).The intermediate hepatocytes were OV-6(-)/CK19(-)/CK7(+)/Hepatocyte(+),Occasionally,they were Hepatocyte(-).C-kit(+) cells were located in the periportal region and fibrous septa.Very few c-kit-positive cells were found integrated into bile duct,the others didn’t coexpress OV-6,CK7 and CK19.
Conclusion Human hepatic progenitor cells activation existed in human liver cirrhosis;The phenotype difference of different cell populations indicated that hepatic progenitor cells were at different stages of the proliferation and differentiation. Combination of different HPCs markers were powerful tools to identify and investigate the HPCs.
Part I-3 Expression of c-kit and CK7 in human hepatocellular carcinoma
Objective To investigate the expression of hepatic progenitor cell marker c-kit and CK7, to explore the relationship between the expression and the clinicopathologic features in human hepatocellular carcinoma.
Methods Surgical specimens from 40 cases of human hepatocellular carcinoma and 3 cases of normal liver were investigated by HE and immunohistochemical staining for c-kit,CK7 and CD45.The stages of tumor cell differentiations were assessed.
Results In normal liver c-kit was negative. We found c-kit(+) tumor cells scattered individually among the tumor cells or located in the periphery of tumor nodules in 19 of 40 HCC. Immunostaining for CK7 was detected in 30 of 40 HCC,and the positive extent varied from each other. Two different immunostaining features were observed,There were significant differences in the expression of HBsAg and Anti-HBc between the c-kit(+) HCC and c-kit(-) HCC or CK7(+) and CK7(-) HCC (p<0.05).The c-kit and CK7 expression was closely related to the degree of tumor cell differentiation(p<0.05).
Conclusion Bone marrow derived hepatic progenitor cells were involved in the liver regeneration during the course of the development of HCC.C-kit and CK7 expression may be a prognostic indicator for HCC.
PartⅡParticipation of the hepatic non-parenchymal cells and extracellular matrix in oval cell-mediated liver regeneration
Objective The aim of the study was to elucidate the the interaction between non-parenchmal cells,extracellular matrix components and oval cells during the restitutive process.
Methods we examined the localization of oval cell,Kupffer cells,hepatic stellate cells ,and the extracellular matrix components laminin and fibronectin using the immunohistochemistry and double Immunofluorescent analyis during the proliferation and differentiation of oval cells in 2-AAF/PH rats model.
Results By day 2 after PH small oval cells began to proliferate around the portal area.Most of desmin(+) HSCs and laminin were present along the hepatic sinusoids in the periportal area.Kupffer cells and fibronectin markedly increased in the whole hepatic lobule.From day 4 to 9,oval cells spread further into hepatic parenchyma,closely associated with HSCs,fibronectin and laminin. Kupffer cells admixed with oval cell by day 6 and then decreased in the periportal zone. From day 12 to 15 most of HSCs,laminin and fibronectin located around the nodus as the differentiation of oval cells into small hepatocte nodus,and minority of them were present in the nodus.Kupffer cells were mainly liminted in the pericentral sinusoids.After day 18 the normal liver lobules structures began to recover.
Conclusion There is a close relationship between the non-parenchymal cells,ECM components and oval cells during the restitutive repair. Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.
PartⅢIsolation,identification and cultivation of rat oval cells and expression of telomerase in oval cells
Objective To establish the proliferative model of rat oval cells,explore the methods of isolating,identifying and culturing the oval cells;To detect the telomerase activity and the expression of correlated genes in oval cells,explore the relationship with the proliferation and differentiation of oval cells.
Methods 2-AAF/PH rat model was used to induce the proliferation of oval cells.The dose of 2-AAF was 15mg/kg. Oval cells were isolated by the modified collagenase perfusion, digestion and gradient centrifugation. Electron microscope examination,RT-PCR and Immunofluorescence were adopted to identify oval cells. Immunohistochemistry,RT-PCR and telomerase activity detection kit were used to examin the expression of the isolated oval cells and oval cell line LE6.
Results We have successfully induced the proliferation of oval cells through the 2-AAF/PH rat model. Freshly isolated cells showed a oval nuclei,a small proportion of cytoplasm and a cobblestone appearance.The diameter of oval cells was about 7 to 12μm.Electron microscope examination showed that there were villose protuberances,a high nucleus/ cytoplasm ratio,immature organelles.Immunofluorescence staining and RT-PCR showed that oval cells expressed 0V-6、AFP、CK19、Albumin、C-KIT、CK18、Thy-1. Immunohistochemistry showed that TERT was located in the nuclei of oval cells around the portal areas.As oval cells differentiated into small hepatocytes,the number of the TERT(+) cells decreased significantly.Both of the isolated oval cells and oval cell lines LE6 expressed the TERT and telomerase RNA(TR).Compared with LE6,the expression of TERT and TR in the isolated oval cells were much lower.The telomerase activity decreased gradually as the increase of the oval cells passage.
Conclusion The 15mg/kg 2-AAF/PH model can induce a great quantity of oval cells to proliferate.High purity oval cells were available through the modified collagenase perfusion,digestion and gradient centrifugation. Telomerase activity may be indispensable for maintaining the proliferative and multi-directional differentiation abilities of oval cells.
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