【作者】 卢宏柱；

【导师】 周建华；

【作者基本信息】 华中科技大学 ， 儿科学， 2009， 博士

【摘要】 第一部分乙型肝炎病毒X基因真核表达载体的构建目的：构建乙型肝炎病毒（HBV）X基因与pCI-neo相融合的高效真核表达载体，为进一步研究HBV X基因的功能奠定基础。方法：设计并合成HBV X基因的引物，以HBV DNA阳性血清PCR扩增得到HBV X基因全序列，将X基因连接到真核表达载体pCI-neo，构成pCI-neo-X质粒，然后用酶切图谱分析、PCR检测和扩增产物序列分析等方法，鉴定所构建的真核表达载体。结果：以重组载体为模板扩增得到的片段大小与已知HBV X基因大小相同，酶切也得到目的基因片段，测序结果也显示与已知X基因序列相同。结论：成功构建了HBV X基因的真核表达载体，为进一步研究HBVX基因的功能奠定了基础。第二部分乙型肝炎病毒X基因转染对大鼠肾小球系膜细胞增殖以及肿瘤坏死因子-α、白细胞介素-1β和白细胞介素-6表达的影响目的探讨HBV X基因表达的蛋白（HBx）对体外培养的大鼠系膜细胞表达肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-6及细胞增殖的影响。方法将前面构成的pCI-neo-X真核表达载体，采用脂质体法将pCI-neo-X转染给体外培养的大鼠系膜细胞，Westem blot测定HBx的表达；以半定量反转录（RT）-PCR测定体外培养大鼠系膜细胞TNF-αmRNA、IL-1βmRNA、IL-6 mRNA表达；酶联免疫吸附试验（ELISA）测定培养上清液中TNF-α、IL-1β、IL-6。甲基噻唑基四唑（MTT）法测定细胞增殖。结果转染pCI-neo-X的系膜细胞，HBx在36和48小时表达明显。同时半定量反转录PCR测定显示TNF-αmRNA、IL-1βmRNA、IL-6 mRNA表达量较未转染和转染空载体组明显增高。培养36和48小时后上清液中TNF-α水平在转染pCI-neo-X组、未转染和转染空载体组分别为（185．3±70．7）pg／ml，（41．7±10．3）pg／ml，（44．0±10．1）pg／ml，（140．3±42．1）pg／ml，（43．7±10．6）pg／ml，（33．3±8．6）pg／ml；IL-1β水平分别为（402±60）pg／ml，（182±23）pg／ml，（170±18）pg／ml，（432±58）pg／ml，（181±20）pg／ml，（165±19）pg／ml；IL-6水平分别为（177．0±18．2）pg／ml，（85．7±16．9）pg／ml，（69．7±22．2）pg／ml，（179．7±26．3）pg／ml，（70．0±18．3）pg／ml，（73．0±14．0）pg／ml，pCI-neo-X组与未转染和转染空载体组比较，均有显著性差异（p＜0．05）；细胞增殖在36和48小时pCI-neo-X组最明显，吸光度分别为1．56±0．36，1．69±0．32，与未转染组0．70±0．08，0．74±0．18和转染空载体组0．68±0．18，0．80±0．19比较，有显著差异（p＜0．05）。结论HBx可诱导大鼠系膜细胞高表达TNF-α、IL-1β和IL-6，促进系膜细胞增殖。HBx对系膜细胞的增殖作用可能与TNF-α、IL-1β、IL-6的高表达有关。第三部分乙型肝炎病毒X蛋白通过激活核因子-κB和细胞外调节蛋白激酶上调大鼠系膜细胞表达肿瘤坏死因子-α目的探讨HBV X基因转染大鼠系膜细胞导致细胞增殖及肿瘤坏死因子-α（TNF-α）高表达的细胞外蛋白调节激酶（ERK）信号转导机制。方法将含有HBVX基因的质粒pCI-neo-X转染入体外培养的大鼠系膜细胞，MTT法检测系膜细胞增殖，半定量RT-PCR检测TNF-αmRNA表达，ELISA检测培养上清液中TNF-α表达。Western blot检测HBx、ERK_1/2及p-ERK_1／2表达。分别采用特异性抑制剂U0126阻断ERK_1/2通路，Lactacystin阻断NF-κB通路和SB203580阻断p38MAPK通路，观察培养细胞增殖、TNF-α及其mRNA表达，以不加抑制剂作为对照。结果转染pCI-neo-X后，系膜细胞增殖明显，细胞TNF-αmRNA及上清液TNF-α表达均增高，通过阻断ERK_1／2或NF-κB通路，系膜细胞增殖减弱，细胞TNF-αmRNA及上清液TNF-α表达均下降。而阻断p38MAPK通路对系膜细胞增殖，细胞TNF-αmRNA及上清液TNF-α表达无明显影响。结论转染HBV X基因可促使系膜细胞增殖，细胞TNF-αmRNA及上清液TNF-α表达均增高，这一作用可能部分是HBV X基因表达的蛋白通过激活ERK_1／2以及NF-κB通路信号而实现。而与p38MAPK通路无关。更多还原

【Abstract】 PARTⅠConstruction of hepatitis B virus X gene eukaryoticexpression vectorObjective:To construct a highly effective eukaryotic expression vector pCI-neo withHBV X gene（pCI-neo-X）.Methods:HBV DNA was extracted from the HBV DNApositive serum.HBV X gene was amplified by PCR,and identified by geloseelectrophoresis analysis.The PCR products and the eukaryotic expression vectors pCI-neowere digested by restriction endonucleases,then the digested X gene was inserted into thedigested eukaryotic expression vector.After screening,the positive recombinants werepicked out.PCR analysis and sequencing were made.Results Both vector DNA directsequence and restriction pattern confirmed that the HBV X gene was seccessfully insertedinto pCI-neo vector.Conclusion:The eukaryotic expression vextor pCI-neo has beensuccessfully constructed. PartⅡHBV X gene transfection induces glomerular mesangialcell proliferation and upregulates TNF-α,IL-1βand IL-6 expressionObjectives The aim of this study is to approach the effect of HBx on expression oftumor necrosis factor-α（TNF-α）,interluekin（IL）-1β,and IL-6 in,and proliferation of ratmesangial cell of rat in vitro.Methods Liposome bearing pCI-neo-X was transfected intocultured rat mesangial cell line of the rat in vitro through liposome method.X geneexpression in the cells was assessed by Western blot.The mRNA expression of TNF-α,IL-1βand IL-6 in expression of cultured mesangial cells line was assessed bysemiquantitative RT-PCR.Mesangial cell proliferation was assessed by MTT.Results HBxobviously expressed in cultured mesangial cells line at 36^th and at 48^th hour aftertransfection.The mRNA of TNF-α,IL-1βand IL-6 expression simutanously increased.Thesupernatent TNF-αlevels in supernatents in MC group,MC plus pCI-neo and MC pluspCI-neo-X at 36^th hours after transfection were （41.7±10.3） pg/ml,（44.0±10.1） pg/ml,（185.3±70.7）pg/ml,respectively,and at 48^th hours were（43.7±10.6）pg/ml,（33.3±8.6）pg/ml,（140.3±42.1） pg/ml,respectively;The supernatent IL-1βlevels in MC group,MC plus pCI-neo and MC plus pCI-neo-X were （182±23） pg/ml,（170±18） pg/ml,（402±60） pg/ml,respectively,at 36^th,and （181±20）pg/ml,（165±19）pg/ml,（432±58）pg/ml,respectively,at 48^th;The supematent IL-6 levels in MC group,MC plus pCI-neoand MC plus pCI-neo-X were （85.7±16.9） pg/ml,（69.7±22.2） pg/ml,（177.0±18.2）pg/ml,respectively at 36^th and （70.0±18.3） pg/ml,（73.0±14.0） pg/ml,（179.7±26.3）pg/ml,respectively at 48^th,The expression of TNF-α,IL- 1βand IL-6 in pCI-neo-X groupwere all markedly higher than those in other two groups（all p＜0.05）.The cell proliferationwas also obvious at the same time.Absorbance in transfcted pCI-neo-X cells were1.56±0.36 at 36^th and 1.69±0.32 at 48^th in hour after transfection,which was markedlyhigher than in untrasfected group 0.70±0.08 at 36^th,0.74±0.18 at 48^th in hour or than intransfcted pCI-neo cells 0.68±0.18 at 36^th,0.80±0.19 at 48^th in hour,respectively,all p＜0.05).Conclussion Therefore,we can conclude that HBx can induce expression ofmRNA of TNF-α,IL-1βand IL-6.HBx may play a critical role in the cell proliferationthrough TNF-α,IL-1βand IL-6 high expression.PartⅢHepatitis B virus X protein upregulates tumor necrosisfactor-αexpression of rat mesangial cell line via NF-κB and ERKspathwayObjectives HBx,a 17-kd protein encoded by X gene of HBV,has been shown tofunction as a transcriptional trans-activator of a variety of viral and cellularpromoter/enhancer elements.The aim of the study is to investigate the signal transductionpathway of HBx in glomerular mesangial cell proliferation and TNF-αexpression.Methods PCI-neo-X was transfected into cultured glomerular mesangial cells.Bothmesangial cell proliferation and supematant TNF-αexpression were compared betweentreated or untreated with ERK inhibitor U0126,NF-κB inhibitor lactacystin and p38inhibitor SB203580 respectively.HBx,ERK_1/2 and p-ERK_1/2 expression in glomerularmesangial cells was assessed by Western-blot.TNF-αmRNA expression was assessed bysemi-quantitative RT-PCR.TNF-αlevels in supernatants were measured by ELISA.Mesangial cell proliferation was assessed by MTT.Results HBx expression was found intransfected glomerular mesangial cells,and became prominent at 36 and 48 hours aftertransfection whatever with or without U0126 in culture media.TNF-αmRNA expressionwas significantly decreased in U0126 group compared with U0126-free group.TNF-αlevels in supernatants in pCI-neo-X transfection without U0126 group were （189.0±18.1）pg/ml at 36 hours and （172.3±24.3） pg/ml at 48 hours after transfection,（41.7±10.3） pg/ml.In contract,TNF-αlevels in supernatants with U0126 were （65.6±11.6） pg/ml and （84.0±24.6） pg/ml.The TNF-αlevels in the latter groups showed significantly lower thanthe formers （P＜0.05）.Glomerular mesangial cell proliferation was also partially inhibitedwhen treated with ERKs inhibitor U0126 or NF-κB inhibitor lactacystin,but no effect wasobserved in p38 inhibitor SB203580 treated group at 36 and 48 hours after transfection.Conclussion HBx can induce glomerular mesangial cell proliferation and increaseTNF-αmRNA expression and its protein production.HBx upregulated TNF-αexpressionand induced cell proliferation of glomerular mesangial cell line partly through ERK_1/2signal transduction pathway.更多还原