绿色荧光蛋白标记的D-氨基酸氧化酶基因在人宫颈癌细胞中的表达研究

为了探讨绿色荧光蛋白标记的红色酵母D 氨基酸氧化酶 (DAAO)基因在人宫颈癌细胞 (HeLa细胞 )中的表达及其功能 ,采用基因重组技术构建了含有CMV启动子和EGFP、DAAO基因开放阅读框 (ORF)的真核表达载体 pIRES DAAO。脂质体法转染HeLa细胞 ,荧光显微镜下观察转染细胞中绿色荧光蛋白的表达 ,流式细胞术分析转染效率并筛选荧光阳性细胞 ,命名为HeLa D。以不同浓度的前药D Ala处理HeLa D细胞 ,MTT法检测细胞存活率。结果显示 ,荧光显微镜下可见绿色荧光蛋白在HeLa D细胞中表达 ,流式细胞术成功筛选出HeLa D细胞。前药D Ala能明显杀伤HeLa D细胞。结果表明 ,EGFP可作为报告基因快速筛选DAAO表达载体转染的细胞 ,DAAO/D Ala自杀基因系统可进一步用于肿瘤的基因治疗研究

Expression and Cytotoxicity of EGFP-labeled D-amino Acid Oxidase in HeLa Cells

To observe the expression of R.gracilis D-amino acid oxidase (DAAO) gene labeled by EGFP in HeLa cells and how it mediates cytotoxicity,DAAO cDNA and EGFP gene were cloned into Eukaryotic expression vector pIRES to construct DAAO gene expression vector pIRES-DAAO.HeLa cells were transfected with pIRES-DAAO in vitro.Expression of EGFP gene in HeLa-D cells was observed under a fluorescent microscope.The efficiency of transfection was analyzed by flow cytometry (FCM).The fluorescent cells were screened by FCM and were named HeLa-D.The activity of HeLa-D cells was detected by MTT colorimetric assay after they were treated with D-Ala at various concentrations.The results indicted that expression of EGFP gene in HeLa-D cells was seen under fluorescent microscope and HeLa-D cells were screened by FCM.Apparently,the prodrug D-Ala killed HeLa-D cells.These results demonstrate that EGFP gene can be regarded as a reporter gene to screen the cells transduced with DAAO quickly,and DAAO/D-Ala suicide gene system may prove helpful in gene therapy of cancer.