【作者】 王爱华；

【导师】 马立宪； 王显军；

【作者基本信息】 山东大学 ， 内科学， 2008， 硕士

【摘要】 研究背景乙型肝炎病毒(hepatitis B virus,HBV)是引起严重肝脏疾病的病原体之一。据世界卫生组织报道,全球20多亿人曾感染乙肝病毒,其中约3.5亿人为慢性乙肝病毒感染者。慢性乙肝不易彻底治愈,并随着病情发展可恶化成肝硬化、肝癌。自1965年Blumberg等发现乙型肝炎病毒表面抗原,到1997年病毒全基因组的克隆和测序完成,人们对HBV的基因组成和抗原的结构、生物特性及其生命周期的认识迅速发展。但因缺乏体外增殖系统和易获得的细胞模型,HBV感染肝脏的早期机理研究进展较为缓慢,从而在一定程度上影响乙型肝炎及其相关肝脏疾病的基础和临床研究。由于人的肝组织来源有限,而且HBV病毒粘附以及DNA转录和复制对宿主有严格的限制,先前研究过的细胞模型又各存在其不可避免的缺陷。因此,建立一个近于自然感染状态即既适用于转染又可用血清中病毒颗粒直接感染,接近肝细胞生理状态并能稳定表达HBV抗原的细胞模型是急需的。本研究旨在通过将利用化学诱变剂诱导产生次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)基因突变的HepG2细胞与人原代肝细胞融合,建立杂交细胞株,这个杂交细胞株从细胞基因工程学理论上讲应该具有双亲遗传学特性,即既具有人原代肝细胞对HBV易感的特性又具有HepG2细胞能体外长期传代的特性。并对HBV在该杂交细胞的感染情况进行初步研究。目的建立不同于HepG2细胞及人原代肝细胞的新型杂交细胞株,使该细胞株既能长期携带HBV又能体外传代培养。方法诱导HepG2细胞产生HGPRT基因突变,将筛选出的HGPRT缺陷的HepG2细胞与携带HBV的人原代肝细胞进行融合得杂交细胞,用HAT培养基筛选出异核体杂交细胞,再利用有限稀释法进行克隆,通过核型分析方法鉴定所得细胞。对所得杂交细胞及培养上清液进行HBV DNA和HBsAg、HBeAg的检测。实验同时,用未杂交的HepG2细胞和人原代肝细胞作对照。结果经筛选后,有一杂交细胞株(HepCHLine3)克隆成功。HepCHLine3在形态学上与HGPRT缺陷HepG2细胞相似,能体外传代培养,染色体核型分析示HepCHLine3杂交细胞染色体众数为99条,证明为融合细胞株,含所有来自HepG2细胞和人原代肝细胞的基因数。传代培养的HepCHLine3及其培养上清液用巢式PCR可分别检测到HBV DNA,培养上清液内检测到HBsAg和HBeAg。对照组的HepG2细胞和人原代肝细胞相应结果为阴性。结果提示该细胞携带并分泌HBV DNA。结论该杂交细胞兼具HepG2细胞体外传代和人原代肝细胞对HBV易感的特性,是一新型杂交细胞株,为进一步建立新型HBV感染细胞模型奠定了基础。更多还原

【Abstract】 BackgroundThe hepatitis B virus(HBV), is one of the major pathogens which can cause severe liver disease. It was reported by WHO, globally, more than 2 billion people were infected by HBV. And there are about 350 million chronic carriers of HBV. The chronic hepatitis B can not be cured, What’s more, hepatitis B can deteriorate to cirrhosis and hepatocellular carcinoma with the development of illness. From Blumberg having found HBV surface antigen in 1965 to having completed clone and sequence of HBV DNA genome in 1997, researchers have been making great progresses in constitution of HBV genome, structure and biological traits of antigens, and life circle of HBV. Due to deficiency of exoteric multiplying system and pragmatic cell models, little was known about the early mechanisms of initiation in infection. Consequently, basic and clinical research on hepatitis B and other types of hepatitis concerned were hampered some way. Owing to the finiteness of human hepatic tissue, the restriction of HBV adhesion, DNA transcription and replication host, at the same time prior cell models are all have their inevitable defects, we need to establish a better cell model. This model should be both suitable for transfection and sensitive to virus particle from blood serum. The cell should be similar to human hepatic cell in morphology and can express HBV antigens stably.ObjectiveTo establish a hybrid cell that is different from either HepG2 cell or human primary hepatic cell. This cell carries HBV, and can be serial subcultivation in vitro.MethodsHGPRT defect HepG2 cell, which was induced by 6-MP, was fused with human primary hepatic cell infected by hepatitis B virus. After being screened with HAT, the hybrid cell was cloned through limiting dilution assay. Subsequently, the hybrid cell was identified by karyotype analysis. HBV DNA and HBsAg、HBeAg were examined at different generations. HepG2 and human primary hepatic cell infected by hepatitis B virus were taken as control groups.ResultsThe hybrid cell is similar to HGPRT defect HepG2 cell morphologically and can be subcultured in vitro. The Karyotype analysis results show that the modal chromosome numbers are 99 in the hybrid cells, indicating that the hybrid cells contain all genomic factors from both HepG2 and human hepatic cell. HBV DNA can be detected in the cells and supernates by nest PCR. In the supernates HBsAg and HBeAg can be detected too. Conceivably the hybrid cell possesses the replication a generation in vitro of HepG2 as well as the sensitivity of human primary hepatic cell to HBV, which paves secretion of HBV DNA.ConclusionAs a new hybrid cell line, it has the characteristics of both generation in vitro of HepG2 and sensitivity of human primary hepatic cell to HBV, which paves way for further study of HBV infection cell model.更多还原