转乙型肝炎病毒x基因肝癌细胞株的建立

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 型肝炎病毒（ＨＢＶ）全基因组中主增ＨＢＸ基因，亚克隆至逆转 功体，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ＱＧＹ７７０１，新霉素（Ｇ４１８）筛选，ＰＣＲ与反转录ＰＣＲ（ＲＴ－ＰＣＲ）鉴定。结果 ＰＣＲ法从ＨＢＶ基因

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

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.