【作者】 何颖；

【导师】 孙树汉；

【作者基本信息】 第二军医大学 ， 遗传学， 2008， 博士

【摘要】 DNA疫苗又称核酸疫苗、基因疫苗,是近年来随着基因治疗技术的发展而产生的一种新型疫苗。由于DNA疫苗本身既有类似减毒活疫苗的优点,又有灭活疫苗或亚单位疫苗的安全性,不仅具有预防疾病的作用,同时还具有治疗疾病的作用。所以治疗性核酸疫苗成为近些年研究的重点,并迅速地从治疗传染性疾病的研究扩展到非传染性疾病的研究。感染疾病预防性和肿瘤治疗性DNA疫苗的研究发展很快,而且有相当数量的临床实验正在进行。尽管DNA疫苗得到了快速而广泛的发展,但其总的免疫效果还不尽如人意。有的DNA疫苗在小动物实验中免疫效果很好,在大动物实验中效果欠佳;有的DNA疫苗在动物实验中能起到很好的免疫保护作用,但在临床试验中却不能保护受试者抵抗病原体的攻击。为了能解决DNA疫苗应用中的这些问题,新的用于增强DNA疫苗的研究策略层出不穷,但任何策略都是建立在对疫苗作用机制深刻认识的基础上,而关于DNA疫苗的免疫机制目前尚不十分清楚。为了深入研究DNA疫苗的免疫机制,我们选择了两种疾病展开研究,一种是阿尔茨海默氏病（AD）,另一种是乙肝病毒（HBV）相关的原发性肝细胞癌（HCC）。阿尔茨海默病（Alzheimer’s disease,AD）是一种神经元退行性变疾病,是引起老年期痴呆最主要的原因。众所周知β淀粉样蛋白（Aβ）在脑内沉积形成的老年斑（senile plaque,SP）是引起AD发病最主要的因素,减少Aβ淀粉样蛋白沉积的形成,是预防和治疗AD的一种新措施。继Schenk等人成功的用Aβ疫苗对PDAPP小鼠进行免疫治疗之后,一些动物实验均发现该疫苗可以诱导产生有效治疗浓度的抗Aβ抗体,使Aβ沉淀减少,并改善动物的认知行为学表现。但在临床Ⅱ期试验中出现了中枢神经系统炎症反应和卒中,这些不良反应促使人们对Aβ疫苗的应用和机制更深入地去研究。也使我们研制具有Aβ免疫原性而又不具有其毒副作用的AβDNA疫苗势在必行。我们前期工作中采用Aβ表位疫苗和Aβ全长DNA疫苗免疫小鼠,不与任何佐剂联用时,Aβ全长DNA疫苗与其中一组Aβ表位疫苗能诱发有效的体液免疫,但抗体滴度不理想。考虑到DNA疫苗所诱导的免疫反应强度与基因表达效率（即每个被转染的细胞内抗原基因的表达量）呈正相关,可以尝试通过改变胞内定位,使之分泌到胞外来改变目的蛋白的免疫原性。本实验选用了TPA信号肽序列与Aβ₄₂融合表达,体内实验证明,含有TPA信号肽序列与Aβ₄₂融合基因的DNA疫苗组所诱发的特异性抗Aβ抗体滴度高于仅含Aβ₄₂全长基因DNA疫苗组。在细胞内表达Aβ/GFP和TPA-Aβ/GFP融合蛋白,发现TPA-Aβ在细胞质和胞核中均匀表达并于72h少量集中分布于细胞膜上,提示我们目的蛋白正处于分泌表达状态。进一步实验证实了在融合基因TPA-Aβ/GFP转染细胞的培养上清液中发现存在目的蛋白。上述实验结果提示我们,利用TPA信号肽成功地将Aβ引导穿过细胞膜,达到分泌表达,从而增强抗原递呈细胞的摄取,提高机体免疫系统免疫活性,产生较高滴度的抗体水平。为我们下一步改造Aβ表位疫苗,使之具有更强的免疫原性,诱导机体产生更强的体液免疫,进一步探讨利用DNA疫苗主动免疫清除老年斑的作用机制奠定了基础。乙型肝炎病毒（hepatitis B virus,HBV）感染和原发性肝细胞癌（hepatocellularcarcinoma,HCC）相关率高达80%,HBV感染者发生HCC的危险性是无感染者的200余倍。HBV是HCC的重要诱因这一结论已被科学界广泛接受,但其具体机制尚不明确。近年来,随着对HBx生物学功能研究的深入,人们发现HBx在此过程中发挥了重要作用。X基因是HBV基因组最小的开放读码框,它编码的X蛋白含154个氨基酸,分子量约为16.5 kD。HBx是一种多功能的病毒调节因子,大量研究资料显示HBx参与基因转录,DNA修复,活化多种信号传导通路。这些效应以及它们在细胞凋亡和增殖中综合作用的结果,可以初步解释HBV导致HCC的机制。但HBx促癌变的确切机制至今尚未阐明,可能通过多种途径起作用,十分复杂,一些研究结果又互相矛盾,因此,有必要对其分子机制进行深入研究,在此基础上才有可能构建相应的治疗性DNA疫苗防治这种与病毒相关的肿瘤。X蛋白不能同双链DNA直接结合,而是通过细胞内蛋白间的相互作用启动一系列磷酸化和去磷酸化过程,从而上调众多基因的表达。为了寻找并鉴定X蛋白直接或间接作用的蛋白,进一步阐明其机理,本实验以成人肝癌细胞HepG2为研究对象,将重组质粒pEGFP-N1-X瞬时转染入细胞中,利用蛋白质组学的核心技术寻找并鉴定了实验组和对照组的8个差异表达蛋白。其中,上调表达的蛋白STRAP、nm23-H1和下调表达蛋白PIMT与PI3K信号通路相关。通过我们进一步的实验发现HBx下调PIMT表达是通过上调STRAP、nm23-H1激活PI3K信号通路实现的。这些结果为我们进一步了解受HBx影响的纷繁复杂的信号通路网络提供一定的帮助,而HBx介导的PIMT低水平表达将为阐明HCC发生发展分子机制提供线索,并为进一步治疗HCC提供靶点。更多还原

【Abstract】 Deoxyribonucleic acid vaccine(DNA vaccine),which is also named nucleic acid vaccine or gene vaccine,is a novel vaccine developing with gene therapy technology in recent years.DNA vaccine not only has advantages of live attenuated vaccine,but also has safety of inactivated vaccine or subunit vaccine,and has both preventive effection and therapeutic effection.Therefore,DNA vaccine for therapy is the key point of research in recent years,and rapidly expands from the treatment of communicable disease to the treatment of the non-communicable disease.Recently,DNA vaccines have entered into a variety of human clinical trials for vaccines against various infectious diseases and for therapies against cancer.Despite their rapid and widespread development,however,the disappointing potency of the DNA vaccines in humans underscores the challenges encountered in the efforts to translate efficacy in preclinical models into clinical realities. In order to improve the efficacy of DNA vaccines,a lot of novel strategies have been performed.The definition of molecular mechanism of DNA vaccine must lead to gain more effective DNA vaccine approaches.However,the mechanisms of immune responses for DNA vaccines are still not entirely understood.In order to improve understanding of the immune response mechanisms,two diseases,Alzheimer’s disease and HBV-related hepatocellular carcinoma,suggesting the development of DNA vaccine may be possible, are investigated in present study.Alzheimer disease(AD) is a neurodegenerative disorder characterized neuropathologically by neuritic plaques-amyloid deposits,neurofibrillary tangles,and selective neuronal loss.The various mouse models and clinical trial results demonstrated that the progressive deposition of Aβhad a central role in AD pathogenesis.Accordingly, Aβhas become a therapeutic target for the prevention and treatment of AD.In 1999, Schenk et al reported that vaccination of an amyloid precursor protein(APP) transgenic mouse model of AD with Aβpeptide resulted in a significant reduction in cerebral Aβdeposits.But this peptide vaccine was complicated by meningoencephalitis among 6%of subjects in phaseⅡa trial.Therefore,the advanced development of AβDNA vaccine having immunogenicity and no toxic,adverse effects is important strategy for AD immunotherapy.In previous study,we have constructed DNA vaccines containing Aβepitope sequences or full-length Aβgene.Although one of Aβepitope encoding plasmid and Aβfull-length encoding plasmid elicited higher Aβ-specific antibody titers than others, the humoral immune responses were not enough satisfied without any adjuvant.According to the positive correlation between antigenic expression levels and the extent of immune activation for DNA vaccine,the immunogenicity of gene would be enhanced by alteration of cellular location and leading product of gene to increased secretion.In the present study, to elevate the immunogenicity of Aβ,the tissue plasminogen activator signal sequence was fused to Aβgene.The results showed that humoral responses were enhanced in mice inoculated with DNA constructs expressing TPA-fused Aβantigen relative to those in mice that had been immunized with AβDNA vaccine.To investigate whether the enhanced immune activation of TPA-fused antigens compared to native protein is the result of increased secretion of protein,pEGFP-N1-Aβand pEGFP-N1-TPA-Aβwere transfected into 293T cells.The fluorescence of TPA-Aβ/EGFP was observed in both the nucleus and cytosol and a few was located at cell membrane at 72h after transfection.This result suggested that TPA- fused protein was secreting from intracellular into extracellular. Western blot analysis confirmed the results by detection of Aβ/EGFP from the cultured supernatant of 293-T-pEGFP-TPA-Aβcells.All of results of experiments in vitro or in vivo demonstrated the targeting antigen was successfully secreted into extracellular conducted by TPA signal sequence.Therefore,we concluded that TPA-fused Aβprotein in host cells should lead to increased secretion of this protein with elevated uptake by antigen-presenting cells,and thus,a more generalized activation of the immune system and the higher levels of specific antibody.In summary,this study provides a clue to apply TPA signal sequence in Aβepitope DNA vaccine and is in favor of elucidating the underlying mechanism of senile plaques clearance by DNA vaccine active immunotherapy in the future research.Chronic HBV infection is closely associated with the incidence of hepatocellular carcinoma.Among the four proteins that originate from the HBV genome,HBx has been reported to be associated with hepatocellular carcinogenesis.The molecular mechanisms of HBx in hepatocellular carcinoma(HCC) are not well understood,since the initiation and propagation of most tumors is a multistep process,and HBx has multiple effects on cellular transcription and the cytoplasmic modulation of signal transfection pathways.Further elucidation of the molecular events that lead to HCC holds promise for improved tumor prevention,and more effective therapies,involving DNA vaccine.HBx does not directly bind DNA but functions via protein-protein interaction.To reveal and identify proteins, interacting with HBx directly or indirectly,which are associated with HCC,the proteomic approach was applied.Eight spots were identified as SEC13L1,PA28a,STRAP,Nm23-H1, APRT,NDUFS3,PIMT and RXRbeta proteins.Among these proteins,one down-regulated pr-otein(PIMT),and two up-regulated proteins(STRAP and nm23-H1),were associated with the PI3K signaling pathway.Furthermore,our experiments showed that PIMT down-regulation by HBx was mediated by the up-regulation of STRAP and nm23-H1, which activated the PI3K/Akt signaling cascade.The altered expression of these proteins could provide more information about very complicated signaling molecular networks activated by HBx protein,and HBx-mediated PIMT down-regulation may provide clues to elucidate the molecular mechanisms of the development and progression of HCC and a target for therapy of HCC.更多还原