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目的 建立表达转乙型肝炎病毒x基因 (HBx)的人肝癌细胞株 ,为研究HBx基因与肝癌生物学行为间的关系提供模型。方法 以聚合酶链反应 (PCR)法从 3.2kb乙型肝炎病毒 (HBV)全基因组中扩增HBx基因 ,亚克隆至逆转录病毒载体 ,磷酸钙共沉淀法将其导入包装细胞系 ,以含病毒的培养上清感染人肝癌细胞系QGY770 1 ,新霉素 (G41 8)筛选 ,PCR与反转录PCR (RT PCR)鉴定。结果 PCR法从HBV基因组扩增出 51 8bp片段 ,序列分析证实其中含HBx基因全编码序列 ;用逆转录病毒将HBx基因转导QGY770 1细胞系 ,G41 8筛选 4～ 6周后出现阳性克隆株QGY/HBx ,PCR法从其基因组中鉴定出全长HBx基因整合 ,RT PCR证实细胞有HBxmRNA的稳定表达。结论 建立了稳定表达HBx基因的肝癌细胞株QGY/HBx
Objective To establish a human hepatoma cell line expressing hepatitis B virus x gene (HBx) and provide a model for investigating the relationship between HBx gene and biological behavior of hepatocellular carcinoma. METHODS: The HBx gene was amplified from the 3.2 kb genome of hepatitis B virus (HBV) by polymerase chain reaction (PCR), subcloned into a retroviral vector, and then introduced into a packaging cell line by calcium phosphate coprecipitation. Virus culture supernatant was infected with human hepatoma cell line QGY770 1, neomycin (G41 8) screening, PCR and reverse transcription PCR (RT PCR) identification. RESULTS: A 516 bp fragment was amplified from the HBV genome by PCR. Sequence analysis confirmed that the HBx gene contained the entire coding sequence; the HBx gene was transduced into the QGY7701 cell line with retroviruses, and positive clones appeared after 4 to 6 weeks of G41 8 screening. QGY/HBx, PCR method identified from its genome full-length HBx gene integration, RT PCR confirmed that cells have a stable expression of HBxmRNA. Conclusion A liver cancer cell line QGY/HBx stably expressing HBx gene was established.