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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- Glu基因表达对肝癌细胞表型和侵袭能力的影响
Objective To establish a hepatoma cell line with high expression of β-glucuronidase (β-Glu) .Methods The recombinant retrovirus expression vector pDOR-βG was transfected into hepatocellular carcinoma cells by liposome-mediated method, G418 positive The expression of β-Glu was observed by immunohistochemistry, enzymatic histochemistry, in situ hybridization and enzymatic activity determination.Results A total of 4 positive clones were obtained, in which F1 and H4 were overexpressed The results of immunohistochemistry showed that the β-Glu immunoreactive substances in transfected cells mainly distributed in the cytoplasm and nucleus, and the enzyme activity of the transfected cells was mainly observed in the cytoplasm by enzyme histochemistry, The transfected cells were in deep color.The positive hybridization signals were observed in the cytoplasm, nucleus and nucleolus by in situ hybridization, and the hybridization signal was enhanced in transfected cells.Conclusion The β-Glu gene can be transferred into human β-Glu to form β- Glu hepatocellular carcinoma high expression model.The establishment of this model can not only be used to observe the gene therapy effect of β-Glu prodrug but also to observe the effect of β-Glu gene expression on the phenotype and invasion ability of hepatoma cells