肝癌细胞内逆转录病毒介导β-葡萄糖醛酸苷酶基因的表达

Expression of β-glucuronidase gene in hepatocellular carcinoma cell line by transfection of retroviral vector

目的 建立高表达 β-葡萄糖醛酸苷酶 (β- Glu)的肝癌细胞株 .方法 采用脂质体介导法 ,将重组逆转录病毒表达载体 p DOR- βG转染肝癌细胞 ,G418筛选出阳性克隆 ,用免疫组化法、酶组织化学法、原位杂交法及酶活性测定等方法观察 β-Glu表达情况 .结果 共获得 4株转染阳性克隆 ,其中 F1,H4为 β- Glu高表达克隆 .免疫组化法发现转染细胞 β- Glu免疫阳性物质主要分布于胞质及胞核内 ,酶组织化学法观察转染细胞酶活性产物主要集中于胞质中 ,且转染细胞较非转染细胞呈色深 .原位杂交法观察阳性杂交信号于胞质及胞核 ,甚至核仁中 ,且在转染细胞中杂交信号增强 .结论 通过转入人β-Glu基因可以建立 β- Glu肝癌高表达模型 .该模型的建立 ,既可用于观察β- Glu前体药物的基因治疗效果 ,也能观察β-

AIM To establish high expression model of glucuronidase(β-Glu) of hepatoma cell line. METHODS A retroviral vector pDOR-βG containing β-Glu cDNA was transfected into hepatoma cell line HCC by lipofectin-transfection methods. After selection and cloning, immunohistochemistry,histochemistry,and in situ hybridization staining etc. were used to measure the expression of β-Glu. RESULTS Several expression clones were obtained. By immunohistochemistry,β-Glu immunoreactivity positive substances were mainly localized in the cytoplasm and nuclei of transfected cells, whilst in HCC cells, that was negative. By histochemistry, β-Glu activity increased significantly in the cytoplasm of HCC-βG cell than that of the control. In situ hybridization histochemistry with digoxigenin labelledβ-Glu cDNA probe showed that mRNA positive signals were located in the cytoplasm and nuclei, even in the nucleoli (hnRNA) of the HCC-βG cells whilst mainly in the cytoplasm in the HCC cells. The β-Glu activity analyzed by biochemistry increased significantly in HCC-βG cells than HCC cells. CONCLUSION β-Glu stable expression cell model could be established by gene transfer method. This model might be used to evaluate the treatment of the prodrug of β-Glu enzyme and to investigate the effect of β-Glu expression on the phenotype of hepatoma cell and invade ability.