乙型肝炎病毒全基因组转基因小鼠的生物学特性研究
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摘要
乙型肝炎病毒(hepatitis B virus,HBV)可引起人类的急、慢性乙型肝炎,而且与肝硬化和肝细胞癌的发生有十分密切的关系,对人类的健康构成了严重的威胁。HBV具有宿主特异性,在自然情况下,HBV不感染医学科学研究中常用的实验哺乳动物——小鼠,80年代中期,由于转基因动物技术的应用,乙肝转基因小鼠模型陆续地在一些发达国家和地区的实验室里出现。由于知识产权的保护以及专利制度等多种因素的限制,我国一直未能引进国外的全基因组乙肝转基因小鼠品系。因此,尽快在我国自行建立一个符合国际标准的、可大规模推广应用的乙肝全基因组转基因小鼠模型是十分必要的。另外,由于HBV有至少4种血清亚型(adr、ayr、adw和ayw),它们在基因组结构、生物学特性和流行地域等方面都存在着一定的差异。在我国流行的主要是adr亚型,所以,我们需要的动物模型应该是相对应的adr亚型的乙肝全基因组转基因小鼠模型。本课题就是利用我们实验室建立并优选的乙肝全基因组转基因小鼠C57-TgN(HBVadr2.0)SMMU“3号”品系的F6-F17代小鼠为实验对象,从DNA、RNA、蛋白质、病毒学、病理学和免疫学角度,综合研究了该品系转基因小鼠的生物学特性,使其成为可应用于乙型肝炎病毒相关研究的实验动物模型。
     本研究采用基因组DNA PCR、Southem blotting和DNA测序的方法,分析了HBV基因在转基因小鼠中的整合特征,结果表明,adr亚型的乙型肝炎病毒全基因组转基因已稳定的整合在转基因小鼠的基因组中,并能通过生殖系以孟德尔遗传方式在世代间稳定遗传。利用RT-PCR和real-time RT-PCR对转基因小鼠肝组织中的总RNA进行定性和半定量检测,分析了HBV基因在转基因小鼠中的转录特征,结果发现,转基因小鼠中整合的HBV基因能够转录出病毒的所有mRNAs,病毒mRNA的半定量检测可用于评价抗病毒治疗的研究,是评价该转基因小鼠的重要指标之一。通过免疫组织化学、血清ELISA和Western blotting方法,分析了HBV基因在转基因小鼠中的表达和分泌特征。肝组织免疫组织化学分析显示,转基因小鼠肝细胞中表达HBsAg、HBcAg和X蛋白三种病毒蛋白,但并不是所有的肝细胞同时表达HBsAg、HBcAg和X蛋白。三种病毒蛋白的检出率分别为:85.82%(HBsAg)、58.25%(HBcAg)和49.61%(X蛋白),HBsAg分布于肝细胞质中;
    
    勺r叼匕洲博d匕学位论文
    乙习跳刀千失病毒全基因启区粉吞因小民的生物学特性减玲究
    HBcAg和x蛋白既可分布于肝细胞核中,也可分布于肝细胞质中。免疫组织化学
    检测可直观的反映转基因小鼠体内病毒蛋白的表达情况,也是评价该转基因小鼠
    的重要指标之一。血清EuSA和认触ste讯blo翻谊g分析发现,转荃因小鼠肝细胞中
    表达的病毒蛋白可分泌入血,小鼠血清中HBSAg和HBeAg的检出率分别为:
    55.20Ok和25.000/0,但HBsAg和HBeAg的存在时间和水平并不同步,具有明显的
    个体差异,另外,转基因小鼠中HBsAg的表达还具有发育调节和性别相关性。采
    用血清DNA PCR、reai一me PCR的方法,分析了转基因小鼠中1{BV的复制、病
    毒颗粒的包装和分泌特征。对小鼠血清DNA进行PCR和real一time PCR分析发现,
    转基因小鼠血清中存在HBV DNA,其含量达到临床患者阳性标准,表明转基因小
    鼠中整合的HBV基因组DNA可复制包装成病毒颗粒并分泌入血。血清中HBV
    DNA的定量检测简便准确,也可用于评价抗病毒治疗的研究,是评价该转基因小
    鼠的重要指标之一。
     我们进一步对该品系转基因小鼠进行了组织病理学、血清酶学和免疫学的检
    测,分析了转基因小鼠的病理学和免疫学特征。肝组织H一E染色分析发现,部分
    转基因小鼠肝组织中出现了不同程度的肝细胞变性(45.52%)、肝细胞坏死区和汇
    管区数量不等的单个核细胞浸润(47.01%)。浸银染色分析发现,29.10%(3 9/134)
    的转基因小鼠肝组织出现了纤维组织增生,特别是单个核细胞浸润处。肝细胞的
    这种病理损伤与转基因小鼠的月龄密切相关,随着月龄的增加,转基因小鼠肝脏
    损伤的发生率和严重程度均增加;另外,肝细胞的变性还与病毒蛋白的表达成负
    相关。透射电镜分析发现,转基因小鼠肝肾脏组织发生了超微病理损伤,主要有:
    线粒体肿胀、线粒体晴消失、内质网扩张、髓样体结构形成、胶原纤维增生。肝
    组织中还有糖原增加、脂滴储积、部分肝细胞溶解等慢性损伤的超微病理改变;
    而肾脏则出现了基底膜增厚、钉突消失、基底膜上出现沉积物、部分细胞核空泡
    样变性等变化。血清酶学分析发现,少数转基因小鼠血清转氨酶(ALT和AST)
    升高。这些结果表明,转基因小鼠发生了与人类慢性HBV无症状携带者相似的肝
    组织病理性损伤,总体为轻度慢性肝炎。采用流式细胞术对该品系转基因小鼠外
    周血T淋巴细胞及其亚群分析发现,转基因小鼠外周血CD犷、CD4+、CDS+T细
    胞和CD4勺CDS+均下降;免疫组织化学检测发现,肝组织中浸润的单个核细胞CD3
    常二二润民吸大学细鹿生物攀欲戈开宜
    
    份晓渊俘创匕学位论文
    乙裂朴炎漪每苦卜刃‘因粗转苏因小且的生物学特性月开究
    和CD4阳性,CDS和CD57阴性:血清ELISA分析发现,转基因小鼠血清A刀ti一HB示
    Anti一HBc、Anti一HBe阴性。
     本课题的研究表明,我们建立的乙肝转基因小鼠C57
Hepatitis B virus (HBV) infection is a serious worldwide public health problem that causes acute and chronic hepatitis. It is also associated with a high incidence of cirrhosis and hepatocellular carcinoma (HCC). Hepatitis B virus has narrow host. In natural condition, HBV could not infect mouse, which is commonly used in medical scientific research. In 1980s, hepatitis B virus transgenic mouse strain was established in some developed countries and areas by transgenic technology. Because of the limiting of intellectual asset and patent, these transgenic mice could not introduce into China yet. It is essential for us to establish a HBV complete genome transgenic mice model ourselves, according with international standard and extending application widely. HBV has at least 4 serosubtypes, ayw, adr, ayr, and adw, among which adr is the most prevailing subtype in China. Therefore we need the HBV transgenic mice harbouring adr subtype genomic DNA. In the current study, we try to study the biological Characteristic
    s including DNA, RNA, protein, virology, pathology and immunology of F6-F17 transgenic mice from transgenic mouse line C57-TgN (HBVadr2.0 ) SMMU 3, which was generated and selected in our laboratory harbouring 2 copies HBV complete genomic DNA (adr subtype).
    In this study, integration of hepatitis B virus gene in transgenic mice genomic DNA was estimated by PCR, Southern blotting and DNA sequencing. Hepatitis B virus genomic DNA was integrated in C57-TgN (HBVadr2.0) SMMU 3 transgenic mice and genetically transmitted from parents to offsprings. Hepatitis B virus mRNAs were evaluated by RT-PCR and real-time RT-PCR using total RNA from transgenic mouse liver tissue. All HBV mRNAs were easily detected in liver tissue. Expression and secretion of virus proteins were estimated by immunohistochemistry, Western blotting and ELISA in transgenic mice. HBsAg, HBcAg and X protein were detected in liver tissue of 85.82% 58.25% and 49.61% transgenic mice respectively by immunohistochemistry. HBsAg was distributed mainly in hepatocytes cytoplasm. HBcAg and X protein were distributed in hepatocytes cytoplasm and/or nuclear of transgenic mice. With Western blotting and ELISA, it was suggested that HBsAg and
    - 6 -
    
    
    HBeAg was secreted into serum in 55.20% and 25.00% transgenic mice. HBsAg level and HBeAg were not parallel in different mice at same development time. And expression of HBsAg was developmental regulation and sexual dimorphism. Hepatitis B virus DNA was detected in serum of transgenic mice by serum DNA PCR and real-time PCR. These results suggested that the hepatitis B virus DNA integrated in transgenic mice could replicate, package up virus particles and secret into serum of transgenic mice.
    We also analyzed the pathological and immunological Characteristics of this transgenic mouse line. Nearly half of them developed chronic liver histopathological lesion including hepatocyte degeneration (45.52%, 61/134), mononuclear inflamed cells infiltrate 47.01% (63/134) in liver tissue by Hematoxylin-Eosin stain. 39 of 134 (29.10%) transgenic mice developed fibers hyperplasia located in portal tract of live by silver stain. It was positive correlation between the frequency and degree of chronic liver lesion and development, and negative correlation between hepatocyte degeneration and expression of virus proteins. Chronic Ultramicro-pathological lesions in liver and kidney were also found including mitochondria swelling, mitochondrial cristae disappearing, endoplasmic reticulum expanding, collagen fibers increasing by ultrahistological analysis. Glycogen enhancing, lipid droplet store and cytolysis were also found in hepatocyte, and basement membrane thickening, immunocomplex depositing on basement membrane and vacuolar degeneration of nuclei were found in kidney. Serum ALT and AST was increased in some transgenic mice. The histopathological changes were similar to human chronic asymptomatic HBV carrier. The CD3\ CD4+, CD8+ T lymphocytes in peripheral blood and CD4*/CD8+ were decreased in transgenic mice by flow cytometry. M
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