RFP标记的YCD基因在HeLa细胞中的表达及其介导的细胞毒性

Expression and Cytotoxicity of RFP-labeled Yeast Cytosine Deaminase in HeLa Cells

背景与目的 :探讨红色荧光蛋白标记的酿酒酵母菌胞嘧啶脱氨酶(YCD)基因在人宫颈癌细胞(HeLa细胞)中的表达及其功能。材料与方法 :采用基因重组技术构建含有CMV启动子和RFP、YCD基因开放阅读框(ORF)的真核表达载体pIRES_YCD ,脂质体法转染HeLa细胞。荧光显微镜下观察转染细胞中红色荧光蛋白的表达 ,流式细胞术分析转染效率及荧光强度并筛选荧光阳性细胞 ,命名为HeLa_Y。以不同浓度的前药5_FC处理HeLa_Y细胞 ,MTT法检测细胞存活率。 结果 :荧光显微镜下可见红色荧光蛋白在HeLa_Y细胞中表达 ,流式细胞术成功筛选出HeLa_Y细胞。前药5_FC能明显杀伤HeLa_Y细胞 ,5_FC浓度与HeLa_Y之间存在对数曲线关系。结论 :RFP可作为报告基因快速筛选YCD表达载体转染的细胞 。

BACKGROUND&AIM: To observe the expression of yeast cytosine deaminase(YCD)gene labeled by RFP in HeLa cells and how it mediates cytotoxicity. MATERIAL AND METHODS: YCD cDNA and RFP gene were cloned into Eukaryotic expression vector pIRES to construct YCD gene expression vector pIRES_ YCD,then HeLa cells were transfected with pIRES_YCD in vitro.Expression of RFP gene in HeLa_Y cells was observed under a fluorescent microscope.The efficiency of transfection and the average intensity of fluorescence were analy...