Hepatic Progenitor Cells/Oval Cells Proliferation In Human Liver Cirrhosis And Hepatocellular Carcinoma, Relationship With Hepatic Microenvironment And Study Of Some Biological Characteristics

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 carcinomaObjective 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 regenerationObjective 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 cellsObjective 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.