【作者】 邵俊斌；

【导师】 陈智；

【作者基本信息】 浙江大学 ， 内科学， 2004， 博士

【摘要】 乙型肝炎病毒(hepatitis B virus，HBV)的生活周期可以分成以下几个阶段：病毒黏附宿主细胞；病毒穿透细胞膜，进入细胞；释放病毒基因组；表达病毒基因产物；复制病毒基因组；组装病毒病毒颗粒；释放病毒。与其它大部分病毒不同，HBV基因组还会从细胞浆穿越核膜进入细胞核。HBV细胞核转位机制包括HBV从胞浆进入胞核和从胞核进入胞浆两个方面，有关这方面的研究已有二十余年，但仍未能形成统一见解。主要的焦点是HBV核衣壳是否进入细胞核。Kamimura，Mc Caul TF、Yamaguchi等通过电镜技术，发现HBV核衣壳可以通过细胞核孔从细胞核内移行到细胞浆中，并且发现核衣壳绝大多数定位在细胞核内，在胞浆中极少。核衣壳从细胞核到细胞浆的转位是通过细胞核孔；转位位点位于细胞核孔中心。与上述报道有分歧的研究认为，核孔直径最大只有25nm,而核衣壳直径在30～35nm，因此核衣壳直接通过核孔的可能性不大，Guiddtti LG等报道核心蛋白转基因小鼠肝细胞中，核衣壳绝大部分定位在细胞核中，只有当肝细胞处于有丝分裂期时，核衣壳才能从核内转到胞浆中，他们研究发现无论从胞核到胞浆，还是从胞浆到胞核，HBV核衣壳均不能穿越。HBV基因组核转位进入细胞核是HBV在肝细胞内复制的重要环节。本研究第一部分采用细胞生物学技术和同位素示踪技术分析HBV核衣壳蛋白、基因组在HBV侵入肝细胞过程中的定位及动态变化，并阐明在HBV穿越细胞核膜过程中，核衣壳蛋白是否需要先蜕去这个问题。 HBV共价闭合环状DNA(covalently closed circular，cccDNA)是HBV复制过程中最为关键的中间体形态。HBV基因组在聚合酶作用下将部分双链开环结构补足成完整双链开环结构，然后在细胞核内连接酶作用下把双链中原先存在的缺口补平，形成共价闭合环状DNA结构，也就是cccDNA。HBV cccDNA在细胞核内RNA聚合酶作用下，转录产生多种RNA，其中中间体RNA是形成子代HBVDane颗粒前提RNA。从病毒生活周期分析，HBV cccDNA是病毒复制以及致病机理的关键所在。此外，HBV cccDNA可与细胞核内的核蛋白结合，形成稳定的微染色体(mini一chromosome)结构，抗病毒药物很难清除它。由于肝细胞往往处在细胞复制周期的G。期，降解HBV cccDNA很难，所以它的自然半衰期就比较长。除HBV基因组整合外，目前临床应用的抗HBV药物和正在研究的治疗乙肝药物的最迫切需要解决的就是消除HBV cccDNA，但是目前没有便捷、准确、灵敏度高的方法可以供临床动态监测HBV cccDNA。Southen blot是检测HBV cccDNA的经典方法，但是该方法过程比较复杂，操作可控性比较差，而且灵敏度不高。Addison等发展了一种能相对定量DHBV的竞争PCR方法，虽然该方法灵敏度有了提高，但是在整个操作过程中仍需进行凝胶电泳、转膜和32P标记的同位素探针杂交。PCR技术是非常灵敏、便捷的方法，但是由于HBV ccoDNA与其它HBV DNA形态非常接近，很难设计出仅仅扩增HBV。 ccDNA而不扩增其它HBV DNA的PCR方法。 本研究第二部分设计了一条特殊的嵌合引物，在该引物的5’端加入与HBV基因组无关的序列，在通过一次单链延伸反应和一次实时荧光定量PCR程序，成功把HBV。ccDNA和非cccDNA形态的其它HBV开环DNA结构区分开，并通过荧光探针杂交，实现高灵敏度、实时定量检测。 论文一乙型肝炎病毒细胞核转位动态监测 一、HBV短期感染细胞制备为研究HBV生活周期中病毒细胞核转位过程，需建立HBV感染细胞模型。HepGZ细胞是人肝癌细胞株，无HBV基因组污染，生长速度快。根据文献报道，在DMSO和一些细胞因子存在下，可以增强对HBV易感性，并且更适合HBV在细胞内复制和表达。 HepGZ细胞长至细胞培养瓶80%面积时，进行细胞收集，并将细胞浓度调节至5 X 10‘细胞/m1，然后以每孔Zml加入到6孔板培养。24小时后，换新培养基(DMEM、5%FCS、1 .5%DMSO、66nM胰岛素、70 pM氢化可的松，l愉用新配)2ml，培养24小时。丢弃上清液，加入HBV DNA阳性血清(HBV DNA载量调节至5 xlo，拷贝/ml)。12小时后，吸弃上清。用DMEM培养基洗涤5次。加入培养基 (DMEM、5%FCS、1 .5%DMSO、66nM胰岛素、70 pM氢化可的松，临用新配)2ml。间隔24小时，吸取上清，并补加新培养基。吸取的上清作HBV相关标志物动态监测。 实时荧光定量PCR法检测培养上清中HBV DNA载量，方法参照说明书进行。放射免疫测定(RIA)培养上清液中HBsAg、HbeAg，操作方法参照说明书进行。免疫组化分析细胞HBcAg表达。HBV DNA阳性血清(5 X IJc叩ies/ml)感染经DMSO、胰岛素、氢化可的松处理的HePGZ细胞后48小时，细胞分泌的HBVDane达到最高值，为8.96x 10污copies/m1，此后逐渐减少，第5天仅为 7.98x10飞oPies/ml，接近本底水平，。细胞分泌的HBv相关抗原也在感染后48小时，达到高峰，HBsAg CPM值最高达1 750，逐渐降低，至第4天已接近临界水平。HBeAg CPM值最高达1 325，逐渐降低，至第4天己接近临界水平。免疫组化结果，在感染细胞中可以找到HBcAg阳性的细胞，在胞浆和胞核均有分布，主要集中在细胞核更多还原

【Abstract】 The life cycle of hepatitis B virus can be divided into several steps: attachment of the virus to the hospital cells; penetration into the cell; release of the viral genome; assembly of virions; release of the virus. In contrast to most other virus, HBV comprises a further pathway, allowing the entry of viral DNA from cytosol into the nucleus. The mechanism of HBV nucleus translocation contains two aspects, one is from cytosol into the nucleus, and another is from the nucleus into cytosol. There still exist contradiction, although many study in this area have been done over past two decade. The focus is whether exists nucleus translocation of core particles. Kamimura T , Me Caul TF ,Yamaguchi et al .found core particles migrated from the nucleus into the cytoplasm through the nuclear pores by electron microscopic observations. Core particles existed in nucleus in most cells. Nucleus translocation site is the mid of nucleus pore. Recent study showed that the maximal diameter of nucleus pore of 25 nm is exceeded by the exprementally-determined size of the core particles of 36 nm by far. The core particles cannot cross the nuclear membrane in ether direction. HBV nucleo-capsids were exclusively in nuclear, but they were released to cytoplasm when nuclear membrane disintegrated during mitosis. The entry of HBV genome to nuclear is an important step of HBV replication in hepatocytes. In the first part of our study, we inspect the core particle protein and genome location and migration during the nucleus translocation by cytobiological technique and isotopes traced method. Inaddition, we clarify the suspicion that nucleo-capsids be dispersed before HBV genome entry to nucleus.Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), the generator of offspring virus particles, is a continuous double chain ring, while other DNA forms contain a gap on each chain at the position of direct repeat 1 sequence (DRl) and direct repeat 2 sequence (DR2) respectively. Once HBV enters cytoplasm, it releases the nucleocapid from the surface antigen envelope; subsequently, the circular genome is translocated to the nucleus. HBV RNA transcription can only occur only after modifications of infecting viral genome. The single strand DNA region of the HBV genome is then converted to double-stranded DNA. Both legations of the nicks that occur in the double strand DNA generate HBV cccDNA. In a sense, cccDNA is the real template of the next generation virus. A 3.5 KB terminally redundant transcript was produced to serve as the pregenomic RNA templates for next generation DNA synthesis through host cellular RNA polymerase II. The minus strand of HBV genome is synthesized by the viral reverse transcriptase. HBV cccDNA appears to be the major viral replicate DNA species; and is resistant to conventional antiviral agents. Lamivudine and interferon are two agents used commonly for treatment of chronic hepatitis B. Lamivudine in particular, is reported to have greatest clinical significance in China. Other studies showed that it could inhibit viral polymerase reverse transcriptase activity, and then repress the production of HBV. It was reported that most patients that continued intaking lamivudine for a period of time, had rapidly falling serum HBV DNA level. However, the decrease of the HBsAg and HBeAg is not as obvious as HBV DNA. The patients’ cccDNA, which synthesizes directly HBV, remaining stable in the hepatocyte nucleus, may account for the resistance of the virus. If the treatment in which lamivudine is discontinued, HBV particles will reappear rapidly so it is difficult to decide how long patients’ treatment with lamivudine should continue. Quantitative analysis of cccDNA is of importance; however, there is still shortage of good methods to detect the HBV cccDNA. Southen blot is a classical method to analyze HBV cccDNA; but, the process is too complex; and the sensitivity is not high developed a quantitativecompetitive PCR assay for DHBV cccDNA. However, in such an assay,electrophoresis and Sou更多还原