【作者】 李常海；

【导师】 裘法祖； 陈孝平；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分转HBx基因大鼠卵圆细胞株的建立及鉴定第一节真核表达质粒pEGF-HBx构建和鉴定目的构建带增强型绿色荧光蛋白(EGFP)的真核表达质粒pEGF-HBx，以利于转染细胞的标记和观察，为进一步研究HBx基因在卵圆细胞中的生物学作用奠定基础。方法用PCR方法从质粒pcDNA3．1-HBx中扩增含KpnⅠ和HindⅢ限制性酶切位点的HBx基因序列。对增强型绿色荧光蛋白质粒载体pEGFP-N1及扩增的HBx目的基因片段进行双酶切，纯化后用连接酶连接得到重组质粒pEGFP-HBx，将其转化大肠杆菌DH5α、涂皿于培养板，卡那霉素筛选培养得到阳性菌落，扩增培养后提取质粒，用双酶切和基因测序鉴定分析质粒是否构建成功。结果双酶切后电泳，显示构建的pEGFP-HBx质粒中含有完整HBx基因大小(465kb)的片段，基因测序显示插入的目的基因连接位点和阅读框架正确，插入序列为完整的HBx基因，说明质粒构建成功。结论成功构建带荧光的真核表达质粒pEGFP-HBx，为转染肝卵圆细胞和进一步研究HBx基因在卵圆细胞中的作用奠定了坚实基础。第二节转HBx基因大鼠卵圆细胞株的建立及鉴定目的建立稳定、高效表达HBx基因的大鼠肝卵圆细胞株，以便进一步研究HBx基因和卵圆细胞在肝癌发生过程中的作用和机制。方法通过脂质体介导法将前面构建的真核表达质粒pEGFP-HBx转染大鼠卵圆细胞(LE／6)，经G418筛选，传代培养后得到稳定表达EGFP-HBx融合蛋白的抗性克隆。分别采用逆转录聚合酶链反应(RT-PCR)和免疫细胞化学(ICC)技术检测抗性克隆中HBx基因的转录和翻译。结果经过一个月左右的筛选培养，获得了稳定表达绿色荧光蛋白的卵圆细胞株；细胞克隆传代培养20代后仍表达强的荧光。RT-PCR及免疫细胞化学检测均发现HBx基因在抗性细胞中得到了稳定转录和翻译。结论pEGFP-HBx质粒能成功地转染卵圆细胞，并在细胞中得到有效复制、转录和翻译。实验获得了稳定、高效表达EGFP-HBx融合蛋白的大鼠卵圆细胞株，为进一步研究转HBx基因卵圆细胞在肝癌发生过程中的作用和机制奠定了基础。第二部分HBx联合AFB1诱导卵圆细胞动物体内转化为肝癌的实验研究第一节HBx联合AFB1诱导肝卵圆细胞裸鼠体内转化为肝癌的研究目的探讨HBx和黄曲霉素(AFB1)是否能诱导肝卵圆细胞异常转化为肝癌，为明确肝癌是否起源于肝卵圆细胞找到直接证据。方法在使用AFB1作为诱癌剂的情况下，分别将转染pEGFP-HBx、pEGFP-N1质粒的大鼠肝卵圆细胞种植于裸鼠肝实质内和皮下，观察种植细胞成瘤情况及性质。实验分为五组：A组：24只，肝内和皮下均种植转HBx基因的卵圆细胞，同时喂食AFB1；B组：20只，肝内和皮下种植转HBx基因的卵圆细胞，不喂食AFB1；C组：20只，肝内和皮下均种植转pEGFP卵圆细胞，同时喂食AFB1；D组：20只，肝内和皮下种植转pEGFP卵圆细胞组，不喂食AFB1；E组：15只，肝内和皮下均不注射细胞，单纯喂食AFB1。用透射电子显微镜和HE染色进行检查；并用免疫组织化学检测肿瘤中HBx、OV6、HepParl、CK8、CK7、PCK、Vimentin、SMA、AFP、C-myc和TGF-α的表达情况，明确其性质。结果16周时，各组皮下成瘤情况为：A组(10／24例)、B组(8／20例)、C组(10／20例)、D组(9／20例)和E组(0／15例)；肝内成瘤情况：只有A组(4／24例)在肝内形成肿瘤，其于各组均为0。皮下肿瘤由间质细地组成，表达OV6、Vimentin和SMA；两种细胞(p-HBx和pEGFP)形成的皮下肿瘤性质相似；透射电镜检查证实皮下肿瘤为不同程度分化的间质瘤。肝内肿瘤由间质性细胞和少数异型性上皮样细胞(癌细胞)组成，后者表达HBx、OV6、HepParl、CK7、CK8、PCK、AFP、C-myc和TGF-α抗原；透射电镜检查发现这些异型性上皮样细胞为癌细胞。因而说明肝内肿瘤为恶性间质细胞瘤和肝细胞癌组成的混合体(肝癌肉瘤)。结论卵圆细胞可在皮下形成间质肿瘤，只有HBx基因和AFB1共同作用才能使肝卵圆细胞在肝内异常转化成肝癌，证明肝癌很可能直接起源于卵圆细胞的异常转化。第二节EMT和MET在肝卵圆细胞转化成肝癌过程中的作用目的研究肝卵圆细胞体内转化成肝癌的机制，探讨上皮细胞-间质转变(EMT)和间质细胞-上皮转变(MET)在这一转化过程中的作用。方法在使用AFB1作为诱癌剂的情况下，将转染HBx基因的大鼠肝卵圆细胞种植于裸鼠皮下，形成皮下肿瘤后再将其移植至肝实质内，使其继续生长，观察成瘤情况和组织学差异。分组为：A组24只，皮下种植转染pEGFP-HBx质粒卵圆细胞；B组20只，皮下种植转pEGFP-N1质粒的卵圆细胞。10周后，选择A组皮下肿瘤10例和B组皮下肿瘤8例，移植至同一裸鼠肝实质内使其继续生长，分组为：C组10只，肝内种植A组(来源于转染HBx细胞)形成的皮下瘤；D组8只，肝内种植B组(来源于未转染HBx细胞)形成的皮下瘤。继续喂食AFB1，观察8周。收集皮下肿瘤和肝内肿瘤，进行HE和透射电镜检查，明确肿瘤性质；同时免疫组织化学染色检测皮下肿瘤和肝内肿瘤中EMT相关标志物CK8、PCK、Vimentin、SMA、E-cadherin和β-catenin的表达差异。结果卵圆细胞种植于皮下后形成完全由间质细胞组成的间质瘤(A和B组)，其强阳性表达Vimentin和SMA，但无E-cadherin和CK表达，表明其发生了EMT。皮下肿瘤移植至肝实质后，可继续生长：来源于转HBx细胞的肝内肿瘤(C组)由占大多数的间质瘤细胞和少数肝癌细胞混杂而成，为肝内癌肉瘤，癌细胞表达HBx、HepParl、CK8、PCK、E-cadherin和β-catenin抗原，而间质瘤细胞中Vimentin和SMA表达下降，表明发生了MET；而未转HBx细胞形成的肝内肿瘤(D组)仍为间质肿瘤，未发生MET。结论不同的局部微环境对卵圆细胞的分化方向起重要作用，在HBx转染和AFB1的共同作用下，肝卵圆细胞可异常转化成肝癌，其转化机制与EMT和MET有关，而且HBx转染很可能促进MET的发生。更多还原

【Abstract】 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.更多还原