脂质体介导CD基因转染SHG-44胶质瘤细胞凋亡的实验研究

目的以含胞嘧啶脱氨酶（CD）基因的pCMVCD重组表达质粒转染SHG-44胶质瘤细胞，体外观察5-氟胞嘧啶（5-FC）对转染CD基因的胶质瘤细胞的凋亡诱导作用。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；脂质体Lipofectamine 2000介导pCMVCD质粒转染SHG-44细胞，G418筛选培养获取抗性细胞克隆（即SHG-44／CD细胞）；免疫细胞化学检测SHG-44／CD细胞的CD基因蛋白水平表达；流式细胞仪、TUNEL实验及透射电子显微镜观察5-FC对表达CD基因的SHG-44／CD细胞凋亡的诱导作用。结果含CD基因的pCMVCD质粒成功转染进入SHG-44细胞，获取了含CD基因的SHG-44／CD细胞，免疫细胞化学染色显示SHG-44／CD细胞成功地表达了CD。在含5-FC的培养液中培养，SHG-44／CD细胞出现典型的凋亡形态，TUNEL显示凋亡细胞比例极高；透射电镜可见凋亡改变；流式细胞术检测凋亡率达18．6％，凋亡率呈剂量依赖性。结论建立了SHG-44恶性人脑胶质瘤细胞的CD／5-FC自杀基因系统。诱导SHG-44／CD胶质瘤细胞产生凋亡可能是脑胶质瘤CD基因疗法的重要机制。

Apoptosis induced by 5-fluorocytosine in SHG-44 human malignant glioma cells genetically modified to express cytosine deaminase

Objective To construct cytosine deaminase/5-fluorocytosine(CD/5-FC) suicide gene therapy system on SHG-44 human malignant glioma cells and investigate whether CD/5-FC system can induce the apoptosis of human malignant glioma cells and the associated mechanism in vitro.Methods The pCMVCD plasmid which includes CD gene fragment was used to transfect SHG-44 cells in vitro using Lipofectamine 2000 cationic lipid reagent.Screened by G418 presence,G418-resistant clones(named SHG-44/CD cells) were isolated.The CD expression in the SHG-44/CD cells were detected by immunohistochemistry.Apoptosis induced by 5-FC in SHG-44/CD glioma cells genetically modified to express cytosine deaminase was investigated by applying Terminal deoxynucleotide transferase-mediated dUTP nick end labeling(TUNEL),electron microscopy and flow cytometry analysis techniques.Results The SHG-44 cells could express CD gene using Liposome-mediated gene transfection.G418-resistant clones(SHG-44/CD cells) were obtained.CD expression in the SHG-44/CD cells were detected by immunohistochemistry.The characters of apoptosis in SHG44/CD cells were observed by TUNEL method and electron microscope respectively.Conclusions CD/5-FC suicide gene therapy system on human malignant glioma cells SHG-44 is successfully constructed.The growth inhibition and apoptosis of SHG-44/CD cells induced by 5-FC in vitro may be a primary mechanism in CD gene therapy for brain glioma,which will play an essential role in glioma gene therapy in vivo.