【作者】 李发武；

【导师】 卢放根；

【作者基本信息】 中南大学 ， 内科学， 2007， 博士

【摘要】 背景HBV感染后的肝病表现有很宽的临床谱，包括无症状携带状态、急性自限性肝炎、慢性活动性肝炎以及暴发性肝炎等。HBV感染者免疫清除病毒的主要效应细胞为CD₈⁺T细胞（CTL）、Th细胞和B细胞，其中CTL介导的细胞特异性免疫是主要途径，决定着感染者的临床结局。免疫因素并不能完全解释HBV感染者复杂的临床转归，HBV变异是另一个影响临床结局的重要因素。近年来，很多HBV变异株被分离和鉴定，并对其生物学特性进行了广泛而深入的研究。核壳蛋白是宿主细胞免疫的核心靶位，在病毒清除和病理损伤过程中起着重要作用，因此HBV／C区基因变异和氨基酸的改变格外引人关注。累计文献和资料，核壳蛋白常见的变异包括P5T、V13A、S35A、L59V、P68L、P130T和P135Q等，这些位点变异对肝细胞损伤无明显影响作用，主要见于无症状携带者，可能是不同地理环境病毒自然进化的结果；核壳蛋白L60V（AA60的亮氨酸被缬氨酸替代）和197L（AA97的异亮氨酸被亮氨酸替代）变异常见于活动性乙型肝炎，L60V、197L变异是否加重肝损伤及其作用机制尚不清楚。通过构建L60V、197L变异HBV全基因表达质粒，转染HepG2细胞，结果表明L60V、197L变异HBV株与野毒株HBV对细胞HLA-Ⅰ表达有不同影响作用；L60V、197L变异HBV全基因重组腺病毒载体注射到小鼠体内的研究表明，L60V、197L变异病毒株更容易免疫逃避。目前关于核壳蛋白功能研究尚少见。野毒株、L60V、197L变异核壳蛋白对细胞HLA-Ⅰ表达的影响作用尚不清楚。HBV全基因组对诱导的细胞凋亡有抵抗作用，但野毒株、L60V、197L变异核壳蛋白对诱导的细胞凋亡是否存在影响尚不清楚。目的研究野毒株核壳蛋白、L60V、197L变异核壳蛋白对HepG2细胞HLA-A表达的影响；研究野毒株核壳蛋白、L60V、197L变异核壳蛋白对诱导HepG2细胞凋亡的影响，通过细胞水平的研究以探讨C区热点变异核壳蛋白与野毒株可能存在的功能差异。方法[1]野毒株C区基因的获得：以P3.8Ⅱ为模板，PCR反应扩增野毒株核壳蛋白基因全长，两端带有EcoRⅠ和BamHⅠ酶切位点。扩增的PCR产物通过琼脂糖凝胶试剂盒回收纯化产物。将纯化的PCR产物与pMD18—T载体连接，转化DH5α克隆扩增，筛选阳性克隆，提取质粒pMD18—HBV／C区基因酶切鉴定。[2]野毒株核壳蛋白表达载体pEGFP-WT的构建：将pEGFP-C1质粒转化扩增，EcaRⅠ和BamHⅠ双酶切，琼脂糖电泳后回收大片段；质粒pMD18—HBV／C区基因EcoRⅠ和BamHⅠ双酶切，琼脂糖电泳后回收小片段。将酶切后的pEGFP-C1质粒与HBV／C区基因进行连接。连接产物转化DH5α克隆扩增，筛选阳性克隆，提取质粒，获得野毒株表达载体pEGFP-WT，酶切和测序鉴定。[3]pEGFP-L97和pEGFP-V60重组载体的获得：通过Quick Change试剂盒在pEGFP-WT的基础上采用特异性引物进行定点突变，PCR反应产物DpnI酶切，转化XL2-Blue超级感受态细胞，铺板挑取突变克隆，筛出新合成的含突变子链的重组载体，酶切测序鉴定。[4]重组载体的转染：用Lipofectamine 2000^TM将pEGFP-C1、pEGFP-WT、pEGFP-V60和pEGFP-L97转染HepG2细胞，6小时后更换为无抗生素的含10％小牛血清的DMEM培养液，24小时后用含G418的培养液筛选。待形成阳性单细胞克隆群落后，用尖吸管吸取单克隆阳性细胞培养，筛选获得阳性细胞培养连续传代3次，建立稳定的细胞株。[5]EGFP-核壳蛋白融合蛋白的鉴定：转染后用荧光显微镜观察有无荧光，以观察转染是否成功。Western blot鉴定核壳蛋白的表达，UVI凝胶成像系统分析条带灰度值，用核壳蛋白／β-actin代表核壳蛋白的相对表达量。[6]HLA-A mRNA和蛋白表达的检测：常规方法提取细胞总RNA，按照RT-PCR试剂盒说明书进行逆转录反应和PCR扩增。经琼脂糖凝胶电泳，用凝胶分析系统对PCR产物进行定量分析。流式细胞术检测HLA-A蛋白的表达。将固定好的细胞每管加入一抗并处理标本，通过流式细胞仪进行检测，每个样品管检测5000个细胞。[7]细胞株凋亡的诱导和检测：经Act-D及TNF-α诱导刺激各组HepG2细胞凋亡，刺激时间16h、32h、48h，使用流式细胞术，利用碘化丙啶染色，检测具有亚G1期DNA含量的细胞比例，代表凋亡细胞数；使用激光共聚焦显微镜，利用Hoechst染色观察32h时各组凋亡细胞核的变化。[8]细胞株凋亡相关通路信号蛋白表达检测：空质粒、pEGFP-WT、pEGFP-V60、pEGFP-L97表达HepG2细胞株培养48h时，提取总蛋白，Western blot检测IKK-α、IκB-α、NF-κB P65、Caspase-8和caspase-3蛋白在各组中的表达。结果[1]PCR成功扩增野毒株核壳蛋白基因；成功将PCR产物连接到pMD18载体；将双酶切后的HBV／C区基因切下并连接到pEGFP-C1上，成功构建野毒株核壳蛋白表达载体pEGFP-WT，酶切和测序鉴定符合实验所需；利用特异性引物成功地在pEGFP-WT基础上实行了定点突变，获得带EGFP与野毒株、L60V、197L变异核壳蛋白的融合蛋白表达重组载体pEGFP-V60和pEGFP-L97，并经测序证实。[2]pEGFP-V60、pEGFP-L97、pEGFP-WT载体和空质粒pEGFP-C1表达细胞株的构建、筛选和鉴定：HepG2细胞的G418最低有效全致死剂量为200gg／mL，确定筛选用浓度为400gg／mL，维持浓度为300μg／mL。通过Lipofectamine转染空质粒、pEGFP-V60、pEGFP-L97和pEGFP-WT进入HepG2细胞，G418筛选2周，挑取阳性细胞单克隆株。荧光显微镜显示EGFP在各细胞系强表达；Western blot检测各细胞系核壳蛋白表达量无明显差别（p＞0.05）。[3]pEGFP-V60、pEGFP-L97、pEGFP-WT和空质粒pEGFP-C1组HepG2细胞株HLA-A mRNA和蛋白表达结果：RT-PCR检测结果表明，空质粒表达HepG2细胞株没有检测到HLA-A mRNA表达，pEGFP-L97组细胞株HLA-A mRNA表达明显高于pEGFP-WT组细胞株，而pEGFP-V60组细胞株HLA-A mRNA表达明显低于pEGFP-WT组细胞株（p＜0.05）。流式细胞术检测表明，pEGFP-V60组细胞株HLA-A表达水平明显低于pEGFP-WT组细胞株，而pEGFP-L97组细胞株HLA-A表达明显高于pEGFP-WT组细胞株（p＜0.05）。[4]流式细胞术检测pEGFP-V60、pEGFP-L97、pEGFP-WT载体和空质粒pEGFP-C1组HepG2细胞株凋亡结果：在16h，32h，48h时，pEGFP-V60、pEGFP-L97和pEGFP-WT组细胞株凋亡率均明显低于空白质粒组细胞株（p均＜0.05）；16h时pEGFP-L97组细胞株凋亡比例明显高于pEGFP-WT组细胞株（p＜0.05），32h时pEGFP-V60组细胞株凋亡比例明显高于pEGFP-WT组细胞株（p＜0.05）。48h时，pEGFP-V60、pEGFP-L97组细胞株的凋亡率均明显高于pEGFP-WT组细胞株（p均＜0.05）。未加刺激因素的各组细胞株在0h时和48h时的凋亡率均无明显变化。[5]激光共聚焦检测细胞凋亡结果：32h时pEGFP-V60、pEGFP-L97组细胞株凋亡比例均明显高于pEGFP-WT组细胞株（p均＜0.05），但低于空白质粒组细胞株（p＜0.05），与流式细胞术的结果相一致。[6]pEGFP-V60、pEGFP-L97、pEGFP-WT载体和空质粒pEGFP-C1组HepG2细胞株凋亡相关信号通路蛋白检测：各组细胞株NF-κB、IKK-α、IκB-α蛋白表达没有明显差别；但pEGFP-WT组细胞株caspase-3，8表达低于pEGFP-V60、pEGFP-L97组细胞株；且三组细胞株caspase-3，8表达均低于空白质粒组细胞株。结论[1]我们成功地克隆了EGFP与野毒株核壳蛋白以及L60V、197L变异核壳蛋白融合表达载体；野毒株核壳蛋白以及L60V、197L变异核壳蛋白细胞株HBcAg表达量无明显差异，能够进一步进行各种核壳蛋白功能差异的研究。[2]核壳蛋白能够刺激HepG2细胞HLA-A mRNA和蛋白的表达；与野毒株核壳蛋白相比，L60V变异核壳蛋白具有下调HepG2细胞HLA-AmRNA和蛋白表达作用，而197L变异核壳蛋白具有上调作用。其机制有待进一步研究。[3]野毒株核壳蛋白具有抑制诱导的HepG2细胞凋亡作用；与野毒株核壳蛋白相比，L60V、197L变异核壳蛋白具有促进细胞凋亡的作用。[4]L60V、197L变异核壳蛋白促进HepG2细胞凋亡作用，可能与L60V、197L变异核壳蛋白上调caspase-3，8表达有关。更多还原

【Abstract】 BACKGROUNDHepatitis B virus (HBV) is a major human infectious pathogen.Many HBV carriers will develop chronic liver diseases, includingcirrhosis and primary hepatocellular cancer (PHC). The spectrum ofinfection ranges from fulminant hepatitis, acute self-limited hepatitis, andchronic hepatitis to asymptomatic carder (AsC) state. Studies have alsoshown that the immune response to HBV remains vigorous long after theepisode of acute HBV infection. Main effective cells to eliminate theHBV include CTL, Th and B cells. The cellular immune responsemediated by CTL is the most critical factor and plays a key role todetermine the clinical outcomes. The clinical outcomes of viral infectionresult from the interaction of viruses and their hosts. Over the past decade,increasing attention has been focused on the contribution of variant HBVstrains to the clinical outcomes of HBV infection, and some importantmutations, which display significant biological functions, have beenidentified, but the underlying mechanisms that are responsible for thediversity of clinical syndromes are not fully understood. It is generallyaccepted that HBV is not directly cytopathic for the infected hepatocyte.Instead, the hepatocellular damage and subsequent viral clearance ismediated by immune response. Core gene mutations are epidemiologicallyassociated with hepatitis activity. Several studies have reported that coregene mutations are more frequently detected in patients with fulminant orsevere hepatitis, but less so in patients with acute self-limited hepatitisand asymptomatic carriers. Thus, it has been suggested that mutations inthe core gene are significantly associated with active liver disease.Mounting evidences showed the hot-spot variations in hepatitis B viruscore gene included P5T, V13A, S35A, L59V, P68L, P130T and P135Qand, these common mutations were detected frequently in the HBV occultcarriers. The high frequency of L60V, I97L mutants in core region weredemonstrated in patients with active hepatitis, but the relationshipbetween these two mutant spots and the disease progress remains elusive. In a recent study, I97L, L60V and wild-type replication-competent HBVgenomes transferred into liver cells have different influence on thecellular expression of HLA-Ⅰ. Other evidences show these mutations inthe core gene might change the immune recognition sites of HBcAgthereby eliciting or evading immune clearance. I97L, L60V and wild-typeHBV strains have different influence on the cellular expression of HLA-Ⅰ.Some reports have demonstrated the influence of wild-type HBV strains,envelope protein and core protein on the apoptosis of HepG2 undergoingapoptosis induced by several cytotoxic agents. But compared with thewide-type core protein, whether core proteins with the hot-spot mutantshave the different influence on the cellular expression of HLA-Ⅰand theHepG2 undergoing apoptosis induced by some factors is still elusive. Wechose two hot-spot mutations in the C gene, L60V and I97L, to studytheir effect on the cellular expression of HL-A and the apoptosis incomparison with the wild-type core protein.AIMTo investigate whether the wide-type, L60V and I97L mutant HBcproteins could have different influence on the cellular expression ofHLA-Ⅰ; To study whether the wide-type, L60V and I97L mutant HBcproteins could have different influence on the HepG2 undergoingapoptosis induced by TNF-αand Act-D and further research the possiblesignaling pathway for them to inhibit the occurrence of apoptosis of theHepG2 cells.METHODS[1] The full-length HBV/C gene cDNA was obtained by PCR from atemplate (p3.8Ⅱplasmid containing 1.2 copies HBV gene) and a pair ofprimers were inserted into the enzyme cutting sites. After PCR, the PCRproduct was purified by gel extraction kit and the purified cDNA of HBcgene was cloned using general T-carrier pMD18-T to obtain clonedcarrier pMD18-HBWC, which was then transformed into DH5α, selected,amplified and then verified by PCR and the double enzyme cutting.[2] The construction of the wild-type HBc protein expressionvector(pEGFP-WT): pEGFP-C1 plasmid was digested by doubleenzymes and then purified by gel extraction kit. pMD18-HBV/C was also digested by double enzyme cutting and then HBV/C cDNA was obtainedand purified from pMD 18-T-HBVP22~e by enzyme cutting. We connectedHBV/C cDNA and pEGFP- by T4 ligase, then the product was clonedinto DH5α, and at last the positive clones were selected and verified byPCR, enzyme cutting and sequencing.[3] The construction of the L60V and I97L mutant HBc proteinexpression vector(pEGFP-V60 and pEGFP-97): We achieved these twovectors by site-mutagenesis on base of pEGFP-WT. Point mutations weremade in using QickChange site-directed mutagenesis kit. After PCR, themethylated parental DNA template without mutation was digested withDpnI restriction enzyme. Then the circular, nicked dsDNA wereTransformed into DH5αsupercompetent cells and repaired. The positiveclones transformed with the mutant plasmid were selected from the LBplates and subsequently the plasmid was extracted and verified by PCR,enzyme cutting and sequencing.[4] The transfection of the recombinant vectors: The eukaryoticexpression carriers, pEGFP-C1, pEGFP-WT, pEGFP-V60 andpEGFP-L97, were transfected into HepG2 cell strains by lipofectine2000respectively and then the positive cells were screened by G418.[5] The identification of the fusion EGFP-core proteins: Theexpression of the fusion EGFP-core proteins were verified usingfluorescence microscope observation and western-blot method. Thequantification was made by analytic software.[6] The detection of HLA-A mRNA and protein expression: TotalRNA was extracted by the rountine way and the HLA-A gene expressionwas detected by the reverse-transcriptase PCR. The products wereelectrophoresis by agrose and the results were quantified by the analyticsoftware.The HLA-A protein expression was measured by FlowCytometry (FCM).[7] Detection and induction of the apoptosis of the various HepG2cell strains: The stimulating duration is 16h, 32h and 48h, respectively.The proportion of cells containing sub-G1 DNA was examined by FlowCytometry (FCM) and propidium iodide (PI) staining. This proportion isused to represent the number of apoptotic cells. Laser Scanning Confocal Microscopy and Hoechst staining are carried out to observe the change ofthe nucleus of apoptotic cells in 32h.[8] The detection of the signal proteins in various groups: Wecollected the cells in 48h and detected the signaling proteins containingIKK-α、IκB-α. NF-κB P65、Caspase-8 and caspase-3 by western blotmethod.RESULTS[1] The wide-type HBV/C gene and clone carrier pMD18-HBV/Chave been obtained successfully, pEGFP-WT, containing the wide-typeHBV/C gene and enhanced green fluorescent protein gene, has been alsosuccessfully obtained, pEGFP-V60 and pEGFP-L97vectors aresuccessfully constructed by site-mutagenesis.[2] The G418 lowest lethal agent is 200μg/ml. The cells transfectedwith pEGFP-C1, pEGFP-WT, pEGFP-V60 and pEGFP-L97 vectors werethen selected using G418 at the concentration of 400μg/ml for two weeks,and the single clone selected showed the existence of the EGFP-fusionprotein by the fluorescence microscope observation respectively; and theexpression of the EGFP-wild-type core protein, L60V and I97L coreproteins, was detected by western blot. The quantitation of these threefusion proteins shows no marked difference(p＞0.05).[3] For the expression of the three fusion proteins, EGFP-wide-typecore protein, L60V and I97L core proteins detected by western blot, thereare no marked difference among these groups (p＞0.05)[4] The results of HLA-A mRNA and protein expression in variousgroups: In various groups, the results from RT-PCR show no expressionin the pEGFP-C1 group and obvious expression in the other groups.Compared with the pEGFP-WT group, The expression of HLA-A mRNAin the pEGFP-L97 one is markedly high (p＜0.05), but that in thepEGFP-V60 one is markedly low (p＜0.05). The results from FlowCytometry (FCM) show the same tendency.[5] The results of the apoptosis from various groups induced byTNF-αand Act-D: The results from flow cytometry and PI staining showthat the proportion of cell apoptosis in other three groups is markedly lowrespectively at various time points compared with the pEGFP-C1 group. In 16h, the proportion of cell apoptosis in the pEGFP-L97 group ismarked higher than that in the pEGFP-WT one. In 32h, the proportion ofcell apoptosis in the pEGFP-V60 group is marked higher than that in thepEGFP-WT one. In 48h, the proportion of cell apoptosis in thepEGFP-V60 group or pEGFP-L97 one is marked higher than that in thepEGFP-WT one respectively. The results from the control HepG2 cells,the pEGFP-C1 group, the pEGFP-L97 group and the pEGFP-V60group, which were not induced by any factors, show no difference in Ohand 48h.[6] The results of apoptotic detection by laser scanning confocalmicroscopy: The proportion of cell apoptosis in the pEGFP-L97 orpEGFP-V60 group is markedly high respectively compared with thepEGFP-WT group in 32h, but lower than that in the pEGFP-C1 one.[7] The results of the signaling proteins concerning the inducedapoptosis: The expression of NF-κb, IKK-α, IKB-αproteins are notmarkedly influenced in various groups, but caspase-3, 8 aredownregulated in the pEGFP-WT, pEGFP-L97 and pEGFP-V60 groupscompared with pEGFP-C1 one. The expression of caspase-3 andcaspase-8 is markedly low in the pEGFP-WT group in comparison withthe pEGFP-L97 and pEGFP-V60 ones.CONCLUSIONS[1] The expression vectors of wide-type core protein, L60V and I97Lmutant core proteins are established; The stable cell clones expressingnearly the same quantitation of the three fusion core proteins could beused to further research their functional difference.[2] The core proteins could upregulate the cellular expression ofHLA-A mRNA and protein. Compared with the wild-type core protein,The expression of HLA-A mRNA and protein in the I97L one is markedlyupregulated, but that in the L60V one is markedly downregulated and themechanism is still elusive.[3] The core proteins could downregulated the induced apoptosis ofHepG2 cells. The cell clones expressing mutant L60V, I97L core proteinsare prone to apoptosis compared with the one expressing the wild-typecore protein. [4] L60V or I97L core protein upregulates the cellular caspase-3 andcaspase-8 expression, which contributes to the upregulated apoptosis ofHepG2 cells transfected with L60V or I97L core protein.更多还原