The Experimental Study Of Hepatic Oval Cell Lines Transformed Into Liver Cancer Following Transfection With HBx Gene And Treatment With Aflatoxin B1 In Vivo

PartⅠ-1 Construction and Identification of Eukaryotic CellExpression Vector of pEGFP-HBxObjective To designed to construct eukaryotic cell expression vector of pEGFP-HBx.andexplore the roles of HBx gene in oval cell.Methods HBx (hepatitis B virus X gene) cDNA fragment with KpnⅠand HindⅢendoenzyme sites was obtained by using PCR (Polymerase chain reaction) from plasmidpcDNA3.1-HBx.The purified HBx gene fragment was inserted into pEGFP-N1 expressionvector,and the recombinant plasmid pEGFP-HBx was identified by double digestion ofrestriction endonuclease and DNA sequencing analysis.Results The recombinant of pEGFP-HBx has whole HBx gene base and correct readingframe as indicated by restriction endonuclease and DNA sequencing analysis.Conclusions The eukaryotic cell expression vector of pEGFP-HBx was successfullyconstructed.It will be helpful in the further study on the roles of HBx and hepatic oval cell incarcinogenesis of PLC (primary liver cancer). PartⅠ-2 Establishment and Identification of Hepatic Oval Cell linesof Transfection with HBx geneObjective To establish stablely and effectively expressing EGFP-HBx fusion protein rathepatic oval cell lines (LE/6) and explore the roles of HBx gene and hepatic oval cell incarcinogenesis of PLC.Methods LE/6 cells were transfected with recombinant eukaryotic cell expression vetor ofpEGFP-HBx (pEGFP-N1 as control) by lipofectine reagent.Resistant to G418 cells wereselected and cloning.The expression of EGFP-HBx fusion protein in clones were examineddirectly with fluorescence microscope,and these clones were isolated and proliferated.Theexpression of HBx was also detected by RT-PCR analysis and immunocytochemistry.Results 4 weeks after transfection with pEGFP-HBx plasmid,the cell clones expressingEGFP-HBx fusion protein were obtained.RT-PCR analysis and immunocytochemistry shownthat HBx gene was expression in cells transfection with pEGFP-HBx.Conclusions The stablely hepatic oval cell strain of transfection with HBx gene andexpressed EGFP-HBx fusion protein was successfully established.It will be helpful in thefurther study the roles of HBx and oval cell in carcinogenesis of PLC. PartⅡ-1 Induced Hepatic oval cell lines Transformation intoLiver Cancer by Transfection with HBx gene and Treatment withaflatoxin B1 in vivoObjective Hepatic oval cells are considered to be the stem cells of the liver,and have alsobeen linked to subsequent development of hepatic malignancies.This study was to investigatewhether hepatic oval cells could been induced transforming into liver cancer by HBx genetransfected and treated with AFB1,and aimed to find direct evidence that liver cancer canarise from hepatic oval cells.Methods The oval cells transfected with plamsid pEGFP-HBx were implanted into the liverparenchyma and subcutaneous of nude mice respectively,treated with and without AFB1.Groups as follows:Group A:24,implanted hepatic oval cells transfected with plasmid p-HBxto the subcutaneous tissue and liver parenchyma,treated with AFB1,Group B:20,implantedhepatic oval cells transfected with plasmid p-HBx to the subcutaneous tissue and liverparenchyma;Group C:20,implanted hepatic oval cells transfected with plasmid pEGFP-N1to the subcutaneous tissue and liver parenchyma,treated with AFB1;Group D:20,implantedhepatic oval cells transfected with plasmid pEGFP-N1 to the subcutaneous tissue and liverparenchyma;Group E:15,no implanted oval cells,only treated with AFB1.The experimentwas terminated at 16 weeks,tumorigenic rate were observed,specimens were collected andparaffinembedded.Routine haematoxylin and eosin (H&E) staining was performed,andtransmission electron microscopy (TEM) was also performed.Immunohistochemistry wasperformed using HBx,OV6,HepPar1,CK8,CK7,PCK,Vimentin,SMA,AFP,C-myc andTGF-α.Results Subcutaneous tumorigenic rate of Group A,B,C,D and E were 10/24,8/20,10/20,9/20 and 0,respectively.These subcutaneous tumor was composited by the mesenchymalcells with expression of OV6,Vimentin and SMA:Two kinds subcutaneous tumor,comingfrom transfected p-HBx cells (Group A and B) and pEGFP-N1 cells (Group C and D),share asimilar histopathological features,There were 4/24 cases of intrahepatic tumor formation inGroup A and no intrahepatic tumor formation in other group.These intrahepatic tumors included components of mesenchymal cells,which accounting for about 80-90% cells andexpressed Vimentin and SMA,and atypical epithelial-like cells,which accounting for about10-20% of cells and expressed HBx,OV6,HepPar1,CK8,PCK,AFP,C-myc and TGF-αantigen.Under TEM.these abnormal epithelial-like cells had distinguished atypia and a fewcell tight junctions between cells could be observed.This result indicated that the atypiaepithelial-like cells were cancerous cells.Therefore these intrahepatic tumors have combinedhistological features of HCC and mesenchymal tumors (i.e.hepatic carcinosarcoma).Conclusions Results from our study provides evidence that the hepatic oval cells can beabnormal transformed,at least partly,into liver cancer on the cooperation of the HBx genetransfection and treatment with AFB1 in liver microenvironment.It is also proposed that theliver cancer can originate from oval cells or progenitor cells. PartⅡ-2 The Role of Epithelial-Mesenchymal andMesenchymal-Epithelial Transition in the process of Hepatic OvalCells lines Transformed into Liver Cancer in vivoObjective This study aim to investigate the role ofepithelial-Mesenchymal transition (EMT)and mesenchymal-epithelial transition (MET) in hepatic oval cells abnormal transformed intoliver carcinosarcoma in vivo.Methods The hepatic oval cells transfection with HBx oncogene (transfected plasmidpEGFP oval cells as control) were implanted into the subcutaneous tissue of nude mice todevelop tumor,treating with AFB1 for 10 weeks.Then the subcutaneous tumors werecollected and divided to transplant into liver parenchyma,at the same time administeringAFB1 for 8 weeks.The subcutaneous tumors and intrahepatic tumors were performed byhaematoxylin and eosin (H&E) and observed by transmission electron microscopy.Immunohistochemistry was performed using HepPar1 antibody as well as EMT-relatedmarkers CK8,PCK,Vimentin.SMA.E-cadherin andβ-catenin.Results 10 weeks after implantion,subcutaneous tumors were formed in the sites ofimplanted oval cells.These tumors consist of mesenchymal cell,expressing of Vimentin andSMA immunoreactivity,but no E-cadherin and CK (cytokeratin) expression.These resultsindicate the occurrence of EMT.The implantation of subcutaneous tumor (arised fromtransfected HBx oval cell) to liver parenchymal formed larger intrahepatic carcinosarcoma.which included some mesenchymal cells areas and some cancerous cells areas were distinctlydifferent from subcutaneous tumors.These intrahepatic tumors showed different degree ofimmunoreactivity for HepPar1,CK8,PCK,E-cadherin andβ-catenin.These results implictedoccurrence of MET.Conclusions Results from our study indicate that the oval cells can be abnormaltransformed into liver cancer on the cooperation of the HBx gene and AFB1,and EMT-METmay play a important role in this process.