转乙型肝炎病毒x基因肝癌细胞株的建立

Establishment of a hepatitis B virus x gene transferred hepatocellular carcinoma cell line

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。

Objective\ To establish a hepatitis B virus x gene transferred hepatocellular carcinoma cell line to provide a model for investigation of the relationship between HBx gene and the biological behavior of hepatocellular carcinoma (HCC). Methods\ HBx gene was amplified from genomic DNA of HBV by PCR technique and subcloned to retroviral vector pLNSX. By using the calcium phosphate DNA co precipitation technique, recombinant plasmid was transferred into packaging cell line and the culture supernatant containing recombinant retrovirus collected to infect the HCC cell line QGY7701. The gene transferred cells were selectively cultured with G418 and identified by PCR and RT PCR techniques. Results\ A 518?bp segment was amplified from HBV genome by PCR, and proved encoding HBx gene by sequence determination. QGY7701 cells with HBx gene transferred were selectively cultured with G418 and the positive clones QGY/HBx obtained in 4 to 6 weeks. A HBx gene integration was detected by PCR in the genomic DNA of QGY/HBx cells and a steady expression of HBx mRNA by RT PCR. Conclusion\ A gene transferred HCC cell line QGY/HBx, which could express HBx gene stably, was established successfully.\;