胞嘧啶脱氨酶自杀基因转染SHG-44胶质瘤细胞的实验表达研究  

Experimental expression study of liposome mediated cytosine deaminase suicide gene transfecting SHG-44 malignant brain glioma cells

作　　者：谢轩贵[1] 游潮[2] 

机构地区：[1]成都市第一人民医院神经外科,四川成都610016 [2]四川大学华西医院神经外科,四川成都610041

出　　处：《中华神经外科疾病研究杂志》2008年第4期309-312,共4页Chinese Journal of Neurosurgical Disease Research

摘　　要：目的以含胞嘧啶脱氨酶(CD)基因的重组表达质粒(recombinant plasmid vector) pCMVCD转染SHG-44胶质瘤细胞,建立胶质瘤嘧啶脱氨酶/5-氟胞嘧啶(CD/5-FC)自杀基因治疗系统。方法体外扩增、酶切鉴定pCMVCD质粒并采用DNA序列测定pCMVCD质粒中的CD基因;改良Eagle培养基(DMEM)培养SHG-44胶质瘤细胞;脂质体lipofectamine2000介导pCMVCD质粒转染SHG44细胞,G418筛选重组子;免疫细胞化学检测表达情况。结果测序证实pCMVCD质粒含有CD基因;脂质体成功介导pCMVCD质粒转染SHG-44细胞,获取了抗性细胞克隆(定名SHG-44/CD细胞);免疫细胞化学染色显示SHG44/CD细胞成功地表达了CD。结论脂质体介导pCMVCD质粒转染SHG-44细胞的方法简单、高效;成功建立了胶质瘤CD/5-FC自杀基因治疗系统,为后续实验研究打下了基础.Objective To transfect SHG-44 malignant brain glioma cells with pCMVCD plasmid contained cytosine deaminase（CD） suicide gene and construct cytosine deaminase/5-fluorocytosine （CD/5-FC） suicide gene therapy system on SHG-44 glioma cells. Methods The pCMVCD plasmid was amplified in Escherichia coli, and purified and cleaved by restriction endonucleases Bam HI and Not I digestion, and identified by electrophoresis. The CD gene fragment was further confirmed by auto-sequencing, The plasmid was transfected into the SHG-44 glioma cells by Lipofectamine 2000-mediated method. Resistant cell clones （SHG-d4/CD） were screened with CA18. CD gene expression on SHG-44/CD cells was evaluated by immunocytochemistry staining. Results DNA sequencing data showed that the pCMVCD plasmid had CD gene fragment. The pCMVCD plasmid was successfully transfected into the SHG-44 glioma cells, and positive cell clones （SHG-dd-/CD）were obtained. Immunocytochemistry staining with CD antibody showed the positive reaction in the cell plasma. Conclusion Lipofectamine 2000 mediated pCMVCD plasmid transfecting SHG-44 glioma cells is simple and efficient. CD/5- FC suicide gene therapy system on SHG-44 glioma cells is successfully constructed.

关 键 词：胞嘧啶脱氨酶 胶质细胞瘤 转染 脂质体 基因表达 

分 类 号：R739.41[医药卫生—肿瘤] Q78[医药卫生—临床医学]