【作者】 许斌；

【导师】 瞿涤； 叶荣； 杨芃原；

【作者基本信息】 复旦大学 ， 病原生物学， 2006， 博士

【摘要】 乙型肝炎病毒(Heptitis B virus，HBV)引起的乙型肝炎是严重危害人类健康的传染病。HBV感染可引起急性或慢性肝炎，并且与原发性肝细胞肝癌的发生密切相关。HBV属于嗜肝DNA病毒科，具有严格宿主特异性，不能感染非灵长类实验动物。目前尚无用于研究HBV感染的细胞培养系统，因此对HBV感染早期阶段的分子机制尚缺乏深入了解，特别是与病毒感染过程相关的细胞蛋白，以及病毒感染对宿主细胞的蛋白表达及功能的影响尚不清楚。鉴于嗜肝DNA病毒具有相似的病毒结构、基因组结构以及复制特点，因此利用其它动物嗜肝DNA病毒如鸭乙型肝炎病毒(DHBV)、土拨鼠肝炎病毒(WHV)等建立的细胞感染模型和动物感染模型，可用于研究乙型肝炎病毒复制和致病机制。在体外培养中，DHBV能够感染原代鸭肝细胞(Primary Duck Hepatocytes，PDHs)、进行有效的复制，感染的细胞可释放具有感染性的病毒颗粒，因此该感染系统对于研究嗜肝DNA病毒复制早期过程具有很高的价值。许多关于HBV复制机理的研究成果便是借助了对DHBV感染模型的研究，如利用DHBV感染模型的研究揭示了HBV复制过程中逆转录策略。以往的研究发现鸭原代肝细胞随着培养时间的延长，细胞对DHBV的易感性逐渐降低，并且逐渐丧失与病毒的结合能力。培养6天的鸭原代肝细胞培养开始表现出对DHBV的易感性降低，培养8天的PDHs对DHBV的易感性仅为培养二天PDHs的10％。然而，当培养基换为含1.5％二甲基亚砜(Dimethyl sulfoxide，DMSO)的无血清培养基时，在培养过程中PDHs对DHBV的易感性降低不明显。我们通过检测不同培养时间和不同培养基条件的原代肝细胞内的病毒核酸和病毒抗原也证实了上述现象。PDHs对DHBV易感性的改变提示在不同的培养时间以及不同培养基条件下培养的细胞内某些蛋白表达发生了变化，从而影响了宿主细胞的结构与功能并导致其对DHBV的易感性发生改变。由于病毒进入宿主细胞具有瞬时性和动态性的特点，因此关于嗜肝DNA病毒感染早期对细胞蛋白表达和功能的影响的研究存在一定的困难。在本研究中我们利用PDHs-DHBV自然感染细胞模型，借助于蛋白组学技术研究与嗜肝DNA病毒早期感染过程相关的宿主肝细胞蛋白以及病毒感染对宿主肝细胞蛋白表达的影响。蛋白质组学技术现已广泛应用于生命科学研究的各个领域，目前已被广泛用于研究各种疾病的发生、发展规律，并取得了很多重大的成就。蛋白质组研究具有观察由多种因素引起的多蛋白质组分整体变化的独特优势，比传统的研究手段具有更多的信息量。蛋白质组学技术的发展推动了病毒在感染中与宿主细胞蛋白相互作用研究的深入，如在HIV、HBV、HCV等多种病毒研究中均借助于蛋白质组学方法发现新的与病毒相互作用的蛋白，为阐明病毒致病机制奠定了基础。对HBV蛋白组学研究主要是集中于HBV X基因对转染细胞、转基因小鼠或病人肝组织蛋白谱的影响研究，以寻找肝癌的早期诊断指标。通过鸭原代肝细胞培养所获的肝细胞纯度高，且可被DHBV自然感染，因此蛋白质组学研究所获得的信息将从整体上反应肝细胞蛋白的变化。鸡全基因组的解密和蛋白组学研究技术的发展，为研究DHBV感染对宿主细胞蛋白表达的影响以及寻找与DHBV易感性相关的细胞蛋白带来了新的契机。目前有关鸭基因的相关研究大多以鸡基因组作为参照，鸭与鸡在进化上亲缘关系较近，本实验室曾根椐鸡的基因序列扩增并克隆了数个鸭cDNA基因(鸭γ-干扰素、鸭α-干扰素、β-actin和GADPH)。因此，利用鸡全基因组公共数据库可以进行有关鸭基因或蛋白的分析和预测，这是我们研究DHBV感染对宿主细胞蛋白表达的影响以及寻找与DHBV易感性相关的细胞蛋白的重要的基础。本论文在建立了PDHs-DHBV感染模型的基础上，利用双向凝胶电泳技术(2-DE)和质谱分析技术，比较分析了对DHBV易感性有差异的鸭原代肝细胞总蛋白，基于鸡全基因组数据库，借助基质辅助激光解析电离飞行时间质谱(matrix assisted laser desorption/ionization-time of flight mass spectrometry)成功鉴定了255个差异蛋白点，并运用生物信息学分析对差异蛋白的功能进行预测与分类。选取差异蛋白中可能与易感性有关的蛋白进行相关基因的克隆及重组表达，并通过细胞内共定位和免疫共沉淀等方法研究了目的蛋白与DHBV表面抗原蛋白的相互作用，分析目的蛋白与易感性改变的关系。第一章鸭原代肝细胞培养方法的建立与优化根据文献报道并结合本实验室条件建立了鸭原代肝细胞的分离培养方法。采用心房灌流法、胸腔动脉灌流法、门静脉灌流法等三种方法进行鸭肝脏原位灌流与肝细胞分离。通过比较上述三种方法优缺点，确定采用经优化的门静脉灌流法进行鸭原代肝细胞的分离。与另外两种方法相比，门静脉灌流法可使肝脏灌流充分、耗用灌流液少、灌流时间短、不易污染，且分离后的PDHs纯度高，分离后的鸭肝细胞于4-6小时可完全贴壁，培养第二天细胞形态呈肝细胞状(细胞呈多角形，胞浆内可见高折光度脂滴)，并开始增殖生长。通过DHBV感染实验，发现所分离培养的PDHs对DHBV易感性较高，培养2、6、8天的PDHs感染病毒后在细胞内均可检测到病毒核酸。病毒感染细胞上清的斑点杂交结果显示PDHs感染DHBV后能释放病毒，证明鸭原代肝细胞可以支持DHBV的自然感染并完成其复制周期。比较培养2、6、8天的PDHs感染DHBV 5天后细胞内和培养上清中的病毒核酸水平，结果显示在培养2天的鸭原代肝细胞对DHBV的易感性最高。第二章对DHBV易感性有差异的鸭原代肝细胞蛋白组学研究利用已建立的PDHs-DHBV感染模型，对不同培养时间及不同培养基条件下培养的PDHs进行DHBV感染实验，利用Southern印迹核酸杂交和免疫组化染色法检测了细胞内病毒核酸和抗原。我们将在含5％胎牛血清(fetal bovin serum，FBS)L-15培养基中培养至第2、4、6、8、10天的PDHs分别感染DHBV，另一组则以含有1.5％DMSO的无血清L-15培养基培养的PDHs，在与上述相同的培养后时间点感染病毒。DHBV感染5天后通过检测细胞内病毒抗原的表达以及病毒DNA在细胞内的复制水平，验证不同培养条件下原代肝细胞对DHBV易感性的差异。实验结果发现，以含5％FBS培养基培养的PDHs随着培养天数的增加对DHBV的易感性逐渐降低，培养10天的PDHs感染DHBV后在细胞内已很难检测出病毒DNA；当培养基换为含1.5％DMSO的无血清培养基时，培养至第10天的PDHs感染DHBV后细胞内病毒核酸才出现较为明显的减少。不同培养条件下培养的PDHs的细胞内病毒抗原检测结果显示：以含5％FBS的培养液培养8天的PDHs感染DHBV后，DHBpreS/S阳性细胞数以及细胞内的病毒表面抗原量明显少于培养2天PDHs感染组，PDHs内病毒抗原显色浅且少，提示在含5％FBS培养基中培养8天的PDHs对DHBV的易感性明显低于5％FBS培养基培养2天的细胞；而用1.5％DMSO无血清培养基培养的PDHs，培养8天与2天的细胞感染DHBV后，细胞内DHBpreS/S阳性细胞数及病毒抗原量相似，提示1.5％DMSO无血清培养基可维持鸭原代肝细胞对DHBV的易感性。在上述研究结果的基础上，我们利用双向电泳技术(2-DE)和质谱分析技术，比较分析了对DHBV易感性有差异的鸭原代肝细胞总蛋白。数据分析的结果显示，在5％FBS培养第2天较第8天有132个蛋白表达发生改变；含1.5％DMSO培养液培养组与含5％FBS培养液培养组比较有123个蛋白改变。在鉴定出的总共255个蛋白中，共有31个为两组中共同改变的蛋白，其中变化一致的有26个。与FBS培养液培养第8天相比均上调的蛋白数目为20个，下调的蛋白数目为6个。结合PDHs对DHBV易感性的实验结果可推测，两组中表达均上调的20个差异蛋白可能与PDHs对DHBV易感性增加有关；两组中表达均下调的6个差异蛋白可能与PDHs对DHBV易感性降低有关。对所鉴定的差异蛋白的生物学功能进行COG(clusters of orthologous genes)分类，预测分析结果显示，多数差异蛋白参与新陈代谢，其余蛋白涉及到信号转导、能量转运、细胞增殖等。从26个差异蛋白中选取可能与病毒感染进程相关的鸭蛋白，以NCBI公布的鸡的相关基因序列为参考设计引物，以鸭mRNA为模板，通过RT-PCR的方法对鸭蛋白基因Ctm4、annexin A2、vinculin、14-3-3 tau、formiminotransferase cyclodeaminase、put．type 5、HPPD等7个基因进行扩增。通过优化PCR条件，成功扩增出鸭Ctm4、annexin A2、14-3-3 tau、HPPD等4种蛋白基因的cDNA。将该4种鸭蛋白基因cDNA序列与Genbank中其他物种相关基因的进行了同源性分析，结果显示，annexin A2、Ctm4、HPPD、14-3-3 tau等cDNA与鸡相应基因的同源性达85.5％-95.9％，氨基酸序列同源性高达97.2％-99.6％。第三章对DHBV易感性不同的鸭原代肝细胞的差异蛋白功能研究在蛋白质组研究结果的基础上，为了分析差异蛋白的相关功能，从26个差异蛋白中选取可能与病毒感染进程相关的鸭蛋白，通过构建相应的重组表达质粒研究其体外表达产物与DHBV感染的关系。鉴于DHBV的表面抗原蛋白DHBpreS/S在病毒感染进程中的重要性，我们研究了上述4种蛋白(annexin A2、Ctm4、HPPD、14-3-3 tau)与DHBpreS/S之间的相互作用。首先将Ctm4、annexin A2、14-3-3 tau、HPPD等4种蛋白cDNA分别克隆至DsRed融合表达载体中(表达鸭相应蛋白的DsRed融合蛋白)，并将DHBpreS/S基因克隆至EGFP融合表达质粒(表达DHBpreS/S的EGFP融合蛋白)。将各重组质粒转染293T细胞后，观察相应融合蛋白在细胞内的表达与定位。之后将4种重组表达质粒分别与DHBpreS/S的EGFP融合表达质粒共同转染293T细胞，观察该4种蛋白与DHBpreS/S在细胞内的共定位情况。细胞内共定位结果显示，annexin A2与HPPD可以与DHBpreS/S在细胞内共定位，其中annexin A2与DHBpreS/S共定位于胞浆细胞膜附近，而HPPD则共定位于细胞核中。为了进一步验证annexin A2与HPPD在细胞内与DHBpreS/S的相互作用，我们构建了表达Myc标签的annexin A2和HPPD重组表达质粒，研究annexin A2和HPPD分别与DHBpreS/S的免疫共沉淀作用。实验结果显示annexin A2与HPPD均可以与DHBpreS/S发生免疫共沉淀，结合细胞内共定位的结果，提示该两种蛋白与DHBpreS/S有相互作用，可能与鸭原代肝细胞对DHBV的感染性有关。第四章感染DHBV前后鸭原代肝细胞差异蛋白的蛋白质组分析由于目前尚无用于研究HBV感染的细胞培养系统，对嗜肝DNA病毒感染宿主细胞后所引起的蛋白表达及功能的影响尚不清楚。本章利用鸭原代肝细胞-DHBV自然感染模型，体外模拟DHBV自然感染过程，运用蛋白组学方法研究分析DHBV感染后PDHs内蛋白表达的改变情况。我们将感染与未感染的鸭原代肝细胞提取总蛋白，进行双向电泳和酶解质谱分析。鉴定后的差异蛋白为127个，其中感染后上调的蛋白有55个，下调的蛋白有72个。差异蛋白的功能预测显示，参与氨基酸转运与代谢的蛋白占13％，参与碳水化合物运输与代谢的占3％，能量产生与转换的相关蛋白占17％，参与信号转导机制的约占13％。亚细胞定位显示差异蛋白以胞浆蛋白为主，约占46％，其次为核内蛋白和线粒体蛋白。DHBV感染后上调的蛋白有膜连蛋白annexinA5、annexin ⅩⅢ、载脂蛋白(apolipoprotein)、中间丝蛋白、14-3-3蛋白、泛素化相关蛋白(26S蛋白酶体亚单位GI28872725)等五十余种，多数蛋白在细胞生命活动中参与细胞信号转导、离子通道、细胞器转运、胞吞胞吐、细胞分泌等过程。我们通过蛋白组学研究发现在DHBV感染的PDHs中APOBEC3G蛋白表达上调，提示病毒的感染诱使宿主细胞的这一抗病毒因子大量表达，发挥其抗病毒效果。抗病毒因子APOBEC在嗜肝病毒感染中的作用机制可能与HIV感染中相似，而嗜肝病毒是否如同HIV一样也存在一个对抗这种拮抗作用的蛋白等都需要通过进一步的实验证明。更多还原

【Abstract】 Hepatitis B virus (HBV) is a major pathogen of acute and chronic hepatitis. HBV infection may result in acute or chronic hepatitis, and may eventually develop to cirrhosis and hepatocelluar carcinomar. HBV belongs to hepadnaviruses family. HBV is primarily hepatotropic virus and only infects human and chimpanzee, but not non-primate laboratory animals. Since lacking of an in vitro infection system, the molecular mechanism of the early steps of HBV life cycle is unclear till now, especially the host proteins related with viral infection as well as effects of viral infections on protein expression of host cell.All members of hepadnaviruses share unique genomic and virion structure features and replicate by using the same strategy through reverse transcription of viral RNA into DNA as an essential step of viral genome replication. The infection models of duck hepatitis B virus (DHBV) and woodchuck hepatitis virus (WHV) are used as alternative system for studying pathogenesis and replication of HBV.It has been reported that the susceptibility of primary duck hepatocytes (PDHs) to DHBV decreased along with the prolonging culture time and PDHs can be maintain its susceptibility to DHBV when culture medium is substituted with fetal bovin serum-free L-15 medium contained 1.5% DMSO. This phenomenon indicating there may be some changes of host protein expression when the PDHs cultured in the different culture conditions, which may affect the susceptibility to DHBV. Since the transient and dynamic property of viral entry, it is difficult to study the early events of viral infection.Nowadays, the proteomic approach is considered to be the key technology in the global analysis of protein expression and in the understanding of gene function in the post-genomic era. By studying global patterns of protein content and activity andhow these change during development or in response to disease, proteomics research has boosted our understanding of systems-level cellular behavior and mechanism of disease. In the present work, we attempted to make comprehensive and comparative analysis of primary duck hepatocytes with different susceptibility to duck hepatitis B virus infection. The differential proteins of PDHs with different susceptibility were identified by using 2-DE and MALDI-TOF-MS/MS to DHBV and the proteins’ functions predicted by using bioinformatics method. We choose some candidate differential protein, amplified their cDNA and cloned into recombinant expression plasmids. Interactions between certain fusion host protein and DHBV surface antigen DHBpreS/S were studied by colocalization in 293T cell line and coimmunoprecipitation.Part I Establishment and modification of duck hepatocytes separation methodAccording to the published reports, we established and modified method of isolating the duck hepatocytes from duckline’s liver to culture primary duck hepatocytes. Three different liver perfusion methods were applied, namely liver was perfused via the cardiac atrium, the thoracic cavity artery, and the portal vein respectively. By comparing these three methods, modified perfusion method via portal vein was adopted. Compared with the other methods, portal vein perfusion method can perfuse the duck liver thoroughly in less time, use less perfusion liquid and decrease the opportunity of bacterial contamination. The isolated duck hepatocytes attached to bottom of cell culture dish in 4 to 6 hours post plating and full extend at the next day.The primary duck hepatocytes could be infected with DHBV naturally. DHBV DNA in the supernatant could be detected by Dot hybridization and intracellular DHBV DNA from DHBV infected PDHs detected by Southern hybridization.Part II Proteomics analysis of primary duck hepatocytes with different susceptibility to DHBVThe susceptibility to DHBV of PDHs growth in different culture medium and at different culture times were studied by using southern hybridization analysis and immunohistochemistry analysis after DHBV infection. The results showed that PDHs may lose their susceptibility to DHBV infection gradually and changes in culture condition may also lead to the susceptibility decrease.To determine whether PDHs’ susceptibility to DHBV infection alters during prolonged culture time, we infected PDHs with DHBV serum at 2 4, 6 8 and 10 days post plating. After incubation overnight with infectious DHBV, PDHs were cultured in L-15 medium with 5% fetal bovin serum (FBS), harvested at day 5 after infection, and total DHBV DNA from PDHs was detected by Southern Blot hybridization with DHBV DNA probe. The highest level of DHBV DNA was detected in infected PDHs at day 2 after plating and intracellular DHBV DNA decreased along with the cultured days, suggesting that PDHs were losing their susceptibility to DHBV infection gradually, that indicated that in 5% FBS culture medium PDHs in the early cultured stage were more susceptibility to DHBV than PDHs cultured for 8 days.In FBS-free L-15 culture medium containing 1.5% DMSO for day 2, 4, 8 after cell plating PDHs showed the same susceptibility to DHBV, but by day 10’s PDHs susceptibility to DHBV decreased. These results suggest that the FBS-free cell culture medium containing 1.5% DMSO maintained the PDHs’ susceptibility to a large extent. The results confirmed by DHBV surface antigen detection of DHBV infected PDHs by immunohistochemistry and immunofluorescence assay.We performed 2D-electrophoresis with proteins of PDHs that have different susceptibility to DHBV infection with differences in culture time and medium. With mass spectra, we found 132 proteins were expressed differently in PDHs at different culture time and 123 in that with different culture medium. Among the 31 proteins shared by the two comparison groups, 26 had consistent correlation with the change of susceptibility. The results indicated these 26 proteins most probably be susceptibility-related to some extent.Part III Functional study of host proteins related with the differential susceptibility of PDHs to DHBV infectionIn order to analyze the functions of host proteins related with the differential susceptibility of PDHs to DHBV infection, total duck liver RNA was extracted and be reverse transcripted into cDNA. Duck Ctm4, annexin A2, 14-3-3 tau and HPPD cDNA eukaryotic expression plasmids were constructed respectively. Since the pivotal role of DHBpreS/S in the process of DHBV infection, we observed the interaction between host proteins and DHBpreS/S using colocalization and coimmunoprecipitation . We constructed recombinant plasmids which express DsRed fusion protein with duck Ctm4, annexin A2, 14-3-3 tau or HPPD and EGFP fusion DHBpreS/S protein. Recombinant plasmids which expressed the duck host fusion proteins were co-transfected with EGFP-DHBpreS/S fusion protein recombinant plasmid into 293T cell line. By Confocal analysis the results showed that duck annexin A2 and HPPD could co-localized in cell plasma with DHBpreS/S. In order to confirm the interaction between host protein and DHBpreS/S, recombinant plasmids expressed the duck host proteins with myc tag were co-transfected with DHBpreS/S expression plasmid. With co-immunoprecipitation analysis, it showed that duck annexin A2 and HPPD could be co-immunoprecipitated with DHBpreS/S, suggesting the both duck proteins can interact with DHBpreS/S and may play the roles in DHBV infection.Part IV Proteomics analysis of primary duck hepatocytes infected and uninfected with DHBVAs no cell model can support HBV natural infection successfully currently, the changes of structure and function of the host cells caused by HBV infection are still unclear. In this part, we infected PDHs with DHBV naturally, then analyzed the protein alteration with proteomics method. After 2-DE and MALDI-TOF-MS/MS,total 127 proteins were identified, which 55 were up-expressed in PDHs infected with DHBV and 72 were down-expressed.Preliminary function prediction showed that 13% of the 127 differentially expressed proteins are involved in amino acid transport and metabolism; 3% are involved in carbohydrate transport and metabolism; 17% are involved in energy production and conversion; 13% are involved in signal transduction mechanisms. Subcell location showed that most of the distinct proteins are in the cytoplasma, the others are mitochondrial proteins and nuclear proteins. We found that anti-HIV infection factor APOBEC3G was up-expressed in PDHs infected with DHBV, which indicated this factor may also serve anti-viral effective on hepadnaviruses infection process.更多还原