【作者】 周健；

【导师】 田德英；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 乙型肝炎是一种常见的传染性疾病，据统计全世界有3.5亿慢性乙型肝炎病毒携带者。慢性乙肝患者发展成为肝硬化的风险比较高，这也是肝细胞癌和非肿瘤性的肝硬化并发症引起较高死亡率的主要原因。目前，治疗慢性乙型肝炎唯一持续有效的方法是系统的IFN-a治疗。但是只有1／3的慢性乙型肝炎患者能获得持续应答。核苷类似物如拉米夫定能使患者血清中的HBV DNA快速下降，也能使患者肝脏病理组织学得到改善。但是治疗停止后常常导致疾病很快复发，而且长期治疗也经常会使病毒发生选择性变异。针对这些情况我们有必要寻找一种新的治疗方法。虽然慢性乙型肝炎发病机制还没有完全被了解，但仍有一个共识，就是肝脏损伤是免疫介导的。具体的免疫治疗办法是尽可能替代使用干扰素或抗病毒药物，以加强治疗慢性乙型肝炎患者的疗效作用。甲基胞嘧啶鸟嘌呤二核苷酸的某些侧序(CpG基序)最早是在细菌DNA内发现的，它对先天性和获得性免疫系统有不同的刺激作用。其中一些作用有助于增强Th1型佐剂抗原特异性反应的活性。例如，CpG DNA能促进95％B细胞的增殖，并分泌免疫球蛋白(Ig)和细胞因子，使B细胞得到保护，避免凋亡，所有这些有助于形成较强的体液反应。CpG DNA也可直接激活单核细胞，巨噬细胞和树突状细胞分泌不同的Th1细胞因子，而这反过来又诱导T细胞和NK细胞分泌更多的细胞因子。总之，CpG诱导产生Th1型细胞因子，主要为白介素-12(IL-12的)和干扰素-g，很少分泌Th2型细胞因子，这些Th1型细胞因子可以使T细胞增强体液和细胞介导的免疫反应。在动物试验中，CpGODN已经被证明与各种抗原联合使用是有效的Th1型疫苗佐剂。例如，通过肌注免疫小鼠注射抗原和CpGODN会产生强烈的细胞毒性T淋巴细胞(CTL)和主要IgG2a抗体，也即Th1型反应。因为Th1型免疫反应被认为是清除乙肝病毒感染必要的反应，CpGODN与重组HBcAg很可能是一种有效的治疗性疫苗，用于治疗慢性乙型肝炎患者。因此，在我们的研究中，首先构建成功含HBcAg和CpG的重组真核表达质粒，肌肉注射重组质粒pZeoSV2(+)／CpG-HBcAg(ISS)免疫BALB／c小鼠，研究其对BALB／c小鼠的免疫作用。同时，将重组质粒pEGFP-N1／CpG-HBcAg(ISS)转染人树突状细胞，研究转染后DC表面分子表达的变化和质粒对DC的免疫影响。此外，将转染DC后的培养上清诱导肝癌细胞株HepG2，研究HepG2的凋亡情况及其机制。最后，研究TLR9在不同病毒滴度的慢性乙肝、丙肝患者外周单个核细胞的表达情况。目的1．构建pZeoSV2(+)／CpG-HBcAg(ISS)和pEGFP-N1／CpG-HBcAg(ISS)基因真核表达载体，为研制乙肝治疗性疫苗奠定基础。2．探讨重组质粒pZeoSV2(+)／CpG-HBcAg(ISS)对BALB／c小鼠免疫的作用。3．探讨重组质粒pEGFP-N1／CpG-HBcAg(ISS)转染人外周血单核细胞来源树突状细胞(DC)后，对其表面分子的表达变化及免疫功能的影响。4．探讨重组质粒pEGFP-N1／CpG-HBcAg(ISS)转染人外周血单核细胞来源树突状细胞后，细胞培养上清诱导肝癌细胞株HepG2凋亡的作用及机制。5．研究TLR9受体在不同病毒载量慢性乙肝、丙肝患者的外周血单个核细胞(PMBC)中的表达。方法1．根据乙肝病毒核心抗原基因序列，设计合成三对引物，在引物中分别引入针对人、鼠敏感的CpG片段和不含CpG的片段，用PCR方法从慢性乙肝病人血清DNA中扩增出乙肝核心抗原基因片段，将扩增的产物与真核表达质粒pZeoSV2(+)和pEGFP-N1连接，构建重组体pZeoSV2(+)／CpG-HBcAg(ISS)和pEGFP-N1／CpG-HBcAg(ISS)，进行酶切、PCR及测序鉴定。2．构建真核表达质粒pZeoSV2(+)／CpG-HBcAg(ISSb，c)，将其免疫BALB／c小鼠，ELISA法检测免疫后小鼠血清中HBcAb、IFN-γ、IL-2、IL-12、IL-4和IL-10的含量。3．构建真核表达质粒pEGFP-N1／CpG-HBcAg(ISSa，c)，将其转染人外周血来源DC。用流式细胞仪检测转染的DC表面CD80和CD86的表达。用ELISA法检测转染后DC培养上清的IFN-γ、IL-12、IL-4和IL-10的水平。4．构建真核表达质粒pEGFP-N1／CpG-HBcAg(ISSa，c)，将其转染人外周血来源DC，用培养上清诱导HepG2的凋亡。检测培养上清诱导HepG2凋亡的变化。5．检测慢性乙肝、丙肝患者血清病毒载量，并分析其与TLR9表达情况的关系。TLR9的蛋白表达水平用流式细胞术检测。研究组包括90名患者(60名慢性乙肝患者，30名慢性丙肝患者)和20名正常健康人作为对照。结果1．乙肝核心抗原基因体外扩增产物大小约为530bp。所构建的pZeoSV2(+)／CpG-HBcAg(ISS)和pEGFP-N1／CpG-HBcAg(ISS)重组体经双酶切及PCR鉴定，与预期片段的大小相符；测序结果与GenBank中收录的HBcAg全长序列一致，表明pZeoSV2(+)／CpG-HBcAg(ISS)和pEGFP-N1／CpG-HBcAg(ISS)真核表达载体构建正确。2．pZeoSV2(+)／CpG-HBcAg(ISSb，c)重组质粒均能诱导BALB／c小鼠产生特异性抗体HBcAb，pZeoSV2(+)／CpG-HBcAg(ISSb)组较pZeoSV2(+)／CpG-HBcAg(ISSc)组产生的抗-HBc效价显著增高(P＜0.01)，且其免疫BALB／c小鼠后能使Th1型细胞因子IFN-γ、IL-2和IL-12的表达增强，抑制Th2型细胞因子IL-4和IL-10的产生。3．pEGFP-N1／CpG-HBcAg(ISSa)转染的DC表面CD80和CD86的表达均有明显升高(P＜0.01)。转染后上清中Th1型细胞因子IFN-γ和IL-12的表达增强(P＜0.01)，Th2型细胞因子IL-4和IL-10的表达下降(P＜0.05)。4．pEGFP-N1／CpG-HBcAg(ISSa)组培养上清对HepG2细胞具有促凋亡作用，随着培养时间延长，细胞凋亡率逐渐增加[0h：(2.8±0.8)％，6h：(7.6±1.0)％，12h：(9.2±1.1)％，18h：(12.6±1.2)％，24h：(17.7±0.7)％，P＜0.05]。5．结果显示与正常对照组比较乙肝、丙肝病毒慢性感染导致TLR9蛋白表达水平减少(P＜0.05)。TLR9蛋白表达水平与慢性乙肝血清病毒载量和慢性丙肝血清病毒载量呈负相关(r=-0.632，r=-0.909，P＜0.01)。结论1．成功地构建了真核重组表达载体pZeoSV2(+)／CpG-HBcAg(ISS)和pEGFP-N1／CpG-HBcAg(ISS)，为CpG的功能研究和乙肝治疗性疫苗的研制提供了物质基础。2．pZeoSV2(+)／CpG-HBcAg(ISSb，c)重组质粒对小鼠HBcAb的产生具有明显的促进作用，含CpG-HBcAg(ISSb)的重组质粒能够明显增强Th1型细胞因子的表达，抑制Th2型细胞因子的表达。3．重组质粒pEGFP-N1／CpG-HBcAg(ISSa)转染可提高DC表面共刺激分子的表达，且能够明显诱导Th1型细胞因子(IFN-γ和IL-12)的产生，抑制Th2型细胞因子(IL-4和IL-10)的产生。4．重组质粒pEGFP-N1／CpG-HBcAg(ISSa)转染培养上清能明显促进肝癌细胞株HepG2的凋亡。5．慢性乙肝、丙肝患者PMBC的TLR9蛋白表达水平降低，且与血清病毒载量呈负相关关系，具有检测病毒复制情况的作用。更多还原

【Abstract】 Hepatitis B virus (HBV) causes a common infectious disease, and there are anestimated 350 million chronic HBV carriers worldwide. Patients with chronic hepatitisB are at high risk of developing liver cirrhosis, and this is associate with a higher rate ofmortality due to the development of hepatocellular carcinoma or noncarcinomatouscomplications of cirrhosis.Currently, the only therapy for chronic hepatitis that has a lasting beneficialeffect is systemic treatment with alpha interferon (IFN-a), but a sustained response isachieved in only one-third of patients with chronic hepatitis B. Nucleoside analoguessuch as lamivudine provide a therapeutic alternative leading to a rapid decrease inserum HBV DNA levels and to histopathological improvement of liver disease.However, cessation of treatment usually leads to a rapid relapse of disease,and longtermtreatment often results in the selection of resistant viral variants. These outcomesemphasize the need for novel therapeutic approaches. Although the pathogenesis ofchronic liver disease is not well understood, there is a consensus that liver damage isimmune mediated. Specific immunotherapeutic strategies have been proposed aspossible alternatives to the use of IFN or antiviral drugs to enhance or to broadenpatients with chronic hepatitis B. Unmethylated cytosine-guanine dinucleotides within the context of certainflanking sequences (CpG motifs), as originally identified in bacterial DNA, have diversestimulatory effects on the innate and adaptive immune systems. Several of these effectscontribute to the strong Th1-type adjuvant activity for antigen-specific responses. Forexample, CpG DNA triggers most (95%) B cells to proliferate, secrete immunoglobulin(Ig) and cytokines, and be protected from apoptosis, all of which contribute to astronger humoral response.CpG DNA also directly activates monocytes,macrophages,and dendritic cells to secrete various Th1 cytokines,which in turn inducesT and NK cells to secrete additional cytokines. Overall, CpG induces a strong Th1-likepattern of cytokine production dominated by inter- leukin-12 (IL-12) and IFN-g, withlittle secretion of Th2 cytokines, and these cytokines can provide additional T-cell helpfor both humoral and cell-mediated immune responses.CpG ODN have been shown tobe effective Th1-type vaccine adjuvants in animals with a variety of antigens. Forexample,mice immunized by i.m. injection of antigen with CpG ODN have strongcytotoxic T lymphocytes (CTL) and predominantly IgG2a antibodies, also indicative ofa Th1-type response. Since such Th1-type immune responses are thought to benecessary tbr clearance of HBV infection,it is possible, that CpG ODN with recombinantHBcAg may be an effective therapeutic vaccine for the treatment of patients chronicallyinfected with HBV.Therefore, in our study, we first constructed eukaryotic expression recombinantvectors for expressing HBcAg and CpG. Then,BALB/c Mouse immunized by i.m.injection of recombinant vectors pZeoSV2 (+) /CpG-HBcAg (ISS) .We explored theimmune effects of pZeoSV2(+)/CpG-HBcAg(ISS) action on BALB/c mice. Meantime,recombinant vector pEGFP-N1/ CpG-HBcAg(ISS) was used to transfect DCs.Then,we have investigated the alteration of the surthce molecules expression on theDCs after transfection and effective to its fucation. In additon,we have investigated theeffects and mechanisms of apoptosis HepG2 induced by DCs culture supernatant,whichwas the DCs after transfected recombinant plasmid pEGFP-N1/CpG-HBcAg(ISS) tohuman monocyte-derived dendritic cells. At last, we have investigated the expression of Toll-like receptors (TLR) 9 in peripheral blood mononuclear cells (PBMC) of patientswith chronic hepatitis B and C of different virus copies infection.Objective1. To construct the recombinant eukaryotic expression vectors pZeoSV2 (+) / CpG-HBcAg(ISS) and pEGFP-N1/CpG-HBcAg(ISS), which provided the basic materialfor the development of Hepatitis B virus remedial DNA vaccine.2. To explore the immune effects of recombinant plasmids pZeoSV2(+)/ CpG-HBcAg(ISS) action on BALB/c mice.3. To explore the alteration of the surface molecules expression on the DCs aftertransfection recombinant plasmid pEGFP-N1/CpG-HBcAg(ISS) to human monocytederiveddendritic cells and effective to its fucation.4. To explore the effects and mechanisms of apoptosis HepG2 induced by DCs culturesupernatant,which was the DCs after transfection recombinant plasmid pEGFP-N1/CpG-HBcAg(ISS) to human monocyte-derived dendritic cells.5. The aim of the present study was to investigate the expression of Toll-like receptors(TLR) 9 in peripheral blood mononuclear cells (PBMC) of patients with chronichepatitis B and C of different virus copies infection.Methods1. Three couples of primers were designed for PCR according to the known sequenceof HbcAg. The CpG fragments sensitive to human or murine and Non-CpG fragmentsare pulled into primers. The HBcAg gene was amplified by PCR from genome ofserumal DNA of chronic HBV patients, and PCR product was subcloned into eukaryoticexpression vector pZeoSV2 (+) and pEGFP-N1. The constructed pZeoSV2 (+) /CpG-HBcAg (ISS) and pEGFP-N1/CpG-HBcAg(ISS) were identified by restrictingenzyme digestion analysis, PCR amplifying, and DNA sequencing.2. Recombinant plasmid pZeoSV2 (+) /CpG-HBcAg (ISSb,c) was used to immuneBALB/c mice. The serum HBcAb, IFN-γ、IL-2、IL-12、IL-4 and IL-10 level weredetected by ELISA.3. Recombinant plasmid pEGFP-N1/CpG-HBcAg(ISSa,c) was constructed and used to transfect DCs.The expression of CD80 and CD86 on the transfected DCs were analyzedby flow cytometry. The IFN-γ、IL-12、IL-4 and IL-10 in the culture supematant of thetransfected DCs were detected by ABC-ELISA.4. Recombinant plasmid pEGFP-N1/CpG-HBcAg(ISSa,c) was constructed and used totransfect DCs. The alteration of apoptosis HepG2 were analyzed by flow cytometry.5. The serum viral load of HBV and HCV was detected in the patients and we analyzedthe correlation between HBV-DNA copies or HCV-RNA copies and the TLR9expression. The protein level of TLR9 was evaluated using flow cytometry. The studygroup was comprised of 90 patients (60 with chronic hepatitis B, 30 with chronichepatitis C) and 20 healthy controls.Results1. The size of amplified HBcAg gene was 530bp. Restriction enzyme digestion, PCRamplifying and DNA sequencing confirmed that pZeoSV2 (+) / CpG-HBcAg (ISS)and pEGFP-N1/ CpG-HBcAg(ISS) had been constructed successfully. Theharvestedfull- length sequence of HBcAg gene was identical with that registered in theGenBank.2. The pZeoSV2 (+) /CpG-HBcAg (ISSb,c) recombinant plasmids can produceHBcAb in BALB/c mice. The anti-HBc titers of pZeoSV2 (+) /CpG-HBcAg (ISSb)groups are obviously higher than those of pZeoSV2 (+) /CpG-HBcAg (ISSc) group(P＜0.01 ) . The result showed that high level of Th1 type cytokines including IL-2,IFN-γand IL-12 were induced in pZeoSV2 (+) /CpG-HBcAg (ISSb) recombinantplasmid groups,where the level of Th2 type cytokines such as IL-4 and IL-10 wereinhibited obviously.3. pEGFP-N1/CpG-HBcAg (ISSa)-transfected DCs expressed higher level of CD80and CD86 (P＜0.01) .The result showed that high level of Th1 type cytokinesincluding IFN-γand IL-12 were induced in pEGFP-N1/CpG-HBcAg(ISSa)recombinantplasmid groups (P＜0.01 ) ,where the level of Th2 type cytokines such as IL-4 and IL-10 were inhibited obviously (P＜0.05) .4..The culture supematant of pEGFP-N1/CpG-HBcAg (ISSa) group increasedapoptosis rate of HepG2 with training time [0h: ( 2.8±0.8 ) % , 6h:(7.6±1.0)%,12h:(9.2±1.1)%, 18h:(12.6±1.2)%,24h:(17.7±0.7)%, P＜0.05].5. Our results demonstrated that HBV or HCV infection led to a decreased expression ofTLR9 protein compared to the healthy group(P＜0.05). The TLR9 protein level isnegative correlation related to serum viral load of HBV and HCV(r=-0.632, r=-0.909, P＜0.01).Conclusions1. The pZeoSV2 (+) /CpG-HBcAg (ISS) has been successfully constructed, whichprovides the basic material for further study the function of CpG and the developmentof Hepatitis B virus remedial DNA vaccine.2. pZeoSV2 (+) /CpG-HBcAg (ISSb,c) has recombinant plasmid an obvious role onHBcAb Production in BALB/c mice. The recombinant plasmids include CpG-HBcAg(ISSb) gene sequence has induced the expression of Th1 type cytokineswherease inhibited the expression of Th2 type cytokines.3. The pEGFP-N1/CpG-HBcAg(ISSa) recombinant plasmid can improve the costimularymolecule on DCs and has induced the production of Th1 type cytokines(IFN-γand IL-12) wherease inhibited the expression of Th2 type cytokines (IL-4and IL-10) .4. The culture supematant of pEGFP-N1/CpG-HBcAg (ISSa) group could significantincrease apoptosis of HepG2.5. There is downregulation of TLR9 protein in PBMC of HBV-infected or HCV-infectedpatients.They are negative correlation related to serum viral load and have an importantrole in detecting viral replication of HBV and HCV.更多还原