【作者】 刘红；

【导师】 张钦宪；

【作者基本信息】 郑州大学 ， 病理学， 2003， 博士

【摘要】 乙型肝炎病毒(Hepatitis B Virus，HBV)严重危害人类的健康，感染机体后可发生急性肝炎、慢性肝炎、肝硬化和肝癌。全球约有3.5亿慢性感染者，亚洲属于高流行区，以围产期传播为主，约有1／3～1／4的慢性HBV感染者将会发生进展性肝病(肝硬化和肝癌)。 对乙型肝炎发病机制的研究一直受到人们的广泛关注。目前认为，乙型肝炎患者肝脏等脏器的病理改变主要是由病毒各基因编码的蛋白与宿主间的免疫反应造成的，但是其具体病理损伤机制还不甚清楚。在HBV各基因编码的蛋白中，HBV中蛋白和核心蛋白均具有较强的免疫原性，能诱导机体产生较强的体液和细胞免疫应答；而且HBV中蛋白和核心蛋白相关的免疫应答成分能否造成肝脏病理损伤还不清楚。为了解乙型肝炎的发病机制，尤其是HBV中蛋白相关的免疫应答成分在乙型肝炎发病中的作用，我们利用已建立的含HBV preS2.S基因的转基因小鼠Balb／c-TgN(preS2-S／ayw)为慢性HBV携带者模型，采用DNA免疫和过继转移的实验方法进行了乙型肝炎发病机制方面的研究。而且，目前对人肝细胞核内核心蛋白的功能还不了解，国内也没有乙肝病毒核心蛋白基因转基因小鼠，为进一步探讨乙肝病毒核心蛋白在乙型病毒性肝炎发病机制中的作用，需建立一个合适的动物模型。本实验分两部分。 第一部分HBV中蛋白相关免疫应答致乙型肝炎转基因小鼠病理损伤的研究 用已建立的含HBV preS2.S基因的转基因小鼠Balb／c-TgN(preS2-S／ayw)与正常同品系小鼠交配，获得127只子代小鼠，经PCR和肝脏活检组织免疫组织化学筛选，得到二者均阳性的转基因小鼠41只。用含HBV preS2.S基因的质郑州人学博l_学位沦义中文摘要粒PcDNA3一S2，S经胫骨前肌肌肉注射免疫正常Balb/c小鼠，获得抗血清和致敏脾细胞。将抗血清、致敏脾细胞(受体小鼠经SGy亚致死剂量照射，以下简称照射)、抗血清十致敏脾细胞(照射)、致敏脾细胞(受体小鼠未经SGy亚致死剂量照射，以下简称未照射)、纯化的单克隆抗体和正常脾细胞经尾静脉注射到含HBv preSZ.S基因的转基因小鼠体内。设同窝非转基因小鼠为对照。在不同时间点(O、2 or4、7、14 or Zld)经眼眶球后静脉采血，检测受体小鼠血清HBsAg、抗HBS抗体、前S2抗原、抗前S2抗体、转氨酶、尿素氮和肌配的变化。并同时颈椎脱臼处死小鼠，取肝、脾、肾、肠、肺和肌肉组织常规石蜡包埋、切片、H一E染色和免疫组织化学检测，观察各器官、组织的病理改变和HBsAg的表达情况。 DNA免疫有效诱导出免疫应答后，将免疫后小鼠抗血清、致敏脾细胞和抗血清+致敏脾细胞分别过继转移到转基因小鼠体内后，转基因小鼠肝脏均不同程度地出现类似人急性乙型肝炎的病理改变，表现为肝细胞广泛的气球样变，血窦内皮细胞肿胀，KuPfl七r细胞增生，汇管区和肝小叶内肝细胞的坏死伴不同程度的单个核细胞的浸润。抗血清组无致敏脾细胞的输入，单个核细胞的浸润出现最早，程度最重，说明肝小叶内浸润的单个核细胞是受体小鼠来源的，是非HBV特异性的，非特异性的免疫应答参与了转基因小鼠肝组织的免疫病理损伤。抗血清+致敏脾细胞组(照射)、致敏的脾细胞组(照射)、致敏的脾细胞组(未照射)三组肝组织的病理变化较抗血清组出现晚，程度较同期为轻，而且致敏的脾细胞组(照射)较致敏的脾细胞组(未照射)病理变化出现晚、程度较同期为轻。经照射的转基因小鼠第7d脾脏萎缩，第14d脾脏增生，镜下可见淋巴细胞增生和多核巨细胞。脾脏的增生与肝小叶内单个核细胞浸润出现的时间是一致的。这些结果进一步的说明肝内浸润的单个核细胞是受体小鼠来源的，可能来源于脾脏，是非抗原特异性的，非特异性免疫应答参与了乙型肝炎的免疫病理损伤。而过继转移正常小鼠的脾细胞没有引起受体小鼠出现类似人急性乙型肝炎的改变，提示特异性免疫应答在乙型肝炎免疫病理损伤中的关键性作用。抗血清+致敏脾细胞 (照射)和抗血清组肾脏有少量单个核细胞的浸润。肺、小肠和肌肉组织等组织无明显的病理改变。对照组小鼠未出现类似人急性乙型肝炎的病理改变，仅有肝细胞的轻微肿胀。转移单克隆抗一HBs抗体和抗前S2抗体导致了肝细胞的广泛水郑州人学博卜，i-一f办_论丈中文摘要肿，但无肝小叶内和汇管区的单个核细胞浸润，两种抗体引起的病理改变无明显差别。而抗血清组除有肝细胞的广泛水肿外，还有肝小叶内单个核细胞浸润，提示单个核细胞浸润是由抗血清中抗体外的成分引起的。抗血清组或/和致敏脾细胞组肝细胞气球样变后有HBsAg表达的减少(P<0.05)，提示气球样变可能是肝细胞清除乙肝病毒的病理表现，肝细胞在乙肝病毒的清除中起着重要作用。 第二部分乙型肝炎病毒核心蛋白基因转基因小鼠的产生 应用受精卵原核显微注射技术，将线性化的含有核心蛋白基因恤Fw亚型)的真核表达质粒peDNA3一HB。注射入C57BU6小鼠受精卵雄性原核中，然后植入12只假孕母鼠。共注射211个受精卵，生产24只仔鼠，全部存活。抽提仔鼠尾组织DNA，PCR筛选到6只Founder小鼠(F。代)，转基因整和率为250/0，与通常25更多还原

【Abstract】 Hepatitis B is one of the most common infectious diseases in the world. Hepatitis B virus (HBV) is a major pathogen of acute hepatitis, chronic hepatitis, cirrhosis and hepatocellular carcinoma. It has been estimated that 350 million people worldwide are chronic hepatitis B virus carriers. The global prevalence of chronic HBV infection varies widely. Asia is the area of the high prevalence of chronic HBV infection. In the areas of high endemicity, perinatal transmission is the main route of transmission. One-quarter to one-third people infected chronically with HBV is expected to develop progressive liver disease (including cirrhosis and hepatocellular carcinoma).Hepatitis B is an important public health issue. There have been many studies on the immunopathgenesis of Hepatitis B. It is well known that the host response to the virus must have a critical role in the pathogenesis of the associated diseases. The precise pathogenetic mechanisms responsible for the various forms of acute and chronic liver diseases are only partly defined. HBV middle protein has stronger immunogenicity. It can induce strong humoral and cell-mediated immune responses. It is unclear whether humoral and cell-mediated immune response related with the middle protein can cause liver lesion. The pathogenesis of hepatitis B and the liver lesion resulted from humoral and cell-mediated immune response in hepatitis B transgenic micewas investigated. Hepatitis B virus core protein, as well as HBV middle protein, can induce stronger humoral and cell-mediated immune responses. It is unclear whether humoral and cell-mediated immune response related with the core protein can cause liver lesion. The function of hepatitis B virus core protein and its interaction with other hepatitis B proteins are unclear. Transgenic model is a useful system to identify the function of core protein and may be an ideal experimental model to investigate the function of core protein. It could provide new insights into core protein. This experiment includes two parts.Part one: Immunopathogenesis of liver lesion resulted from immune response about HBV middle protein in HBV transgenic miceHBV transgenic mice Balb/c-TgN (preS2.S/ayw) were mated with same linage mouse. Mice were screened by PCR and immunohistochemical analysis. To study the pathogenesis of hepatitis B and the liver lesion resulted from humoral and cell-mediated immune response in hepatitis B transgenic mice Balb/c-TgN (preS2.S/ayw) , anti-sera and primed spleen cells were collected from the normal Balb/c mice injected on a single occasion with 100ug recombinant DNA (pcDNA3-S2.S) encoding HBV middle envelope protein . DNA was injected directly into regenerating tibials anterior muscles . An Hepatitis B transgenic mouse lineage Balb/c-TgN (preS2.S/ayw) model system was used to analysis the immunopathogesis of HBV-induced liver disease. Anti-sera and primed spleen cells were collected. And anti-sera> primed spleen cells(recipient mice with 5Gy irradiation), anti-sera+primed spleen cells(recipient mice with 5Gy irradiation), primed spleen cells(recipient mice without 5Gy irradiation), normal spleen cells, anti-HBs antibody and anti-preS2 antibody were passively transferred into transgenic mice (harbouring HBV preS2 and S genes) and their nontransgenic littermates. Blood was collected from anesthetized mice by retrobulbar puncture using heparinized glass pipettes at various intervals (0, 2 or 4, 7, 14 or21 d)after immunization to measure the level of HBsAg, anti-HBs antibody, preS2antigen , anti-preS2 antibody, ALT, AST, Cr and Urea. At meanwhile, the tissues of mice were fixed in formalin. Paraffin embedded tissues were sectioned and stained with hematoxylin/eosin. The expression of hepatitis B surface antigen was assessed by immunohistochemical analysis.The immunization in mice was induced efficiently. After passive transfer of anti-sera, the pathological changes in the liver of the transgenic mice resembled what were observed in human acute hepatitis B. The pathological change of liver was chara更多还原