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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
In order to investigate the expression and function of green fluorescent protein-labeled red yeast D-amino acid oxidase (DAAO) gene in human cervical cancer cells (HeLa cells), we constructed a cDNA library containing CMV promoter, EGFP, (ORF) eukaryotic expression vector pIRES DAAO. HeLa cells were transfected by lipofectamine. The expression of green fluorescent protein (GFP) in transfected cells was observed under a fluorescence microscope. The transfection efficiency was analyzed by flow cytometry and the fluorescent positive cells were screened, named HeLaD. HeLa D cells were treated with different concentrations of prodrug D Ala, and cell viability was determined by MTT assay. The results showed that under the fluorescence microscope, green fluorescent protein was expressed in HeLa D cells, and HeLa D cells were successfully screened by flow cytometry. Prodrug D Ala markedly killed HeLa D cells. The results showed that EGFP could be used as reporter gene to screen DAAO expression vector and DAAO / D Ala suicide gene system could be further used in gene therapy of tumor