论文部分内容阅读
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 ,YCD/5_FC自杀基因系统可以进一步用于肿瘤的基因治疗研究
BACKGROUND & AIM: To investigate the expression of red fluorescent protein-labeled Saccharomyces cerevisiae cytosine deaminase (YCD) gene in human cervical carcinoma cells (HeLa cells) and its function. MATERIALS AND METHODS: The eukaryotic expression vector pIRES_YCD containing CMV promoter, RFP and YCD open reading frames (ORFs) was constructed by gene recombination technique and transfected into HeLa cells by liposome. Fluorescence microscopy was used to observe the expression of red fluorescent protein in transfected cells. The transfection efficiency and fluorescence intensity were analyzed by flow cytometry, and the fluorescent positive cells were screened for the expression of HeLa_Y. HeLa_Y cells were treated with different concentrations of prodrug 5_FC, cell viability was measured by MTT assay. Results: Fluorescent microscopy showed that the red fluorescent protein was expressed in HeLa_Y cells. HeLa_Y cells were successfully screened by flow cytometry. Prodrug 5_FC can significantly kill HeLa_Y cells, 5_FC concentration and HeLa_Y relationship exists between the logarithmic curve. Conclusion: RFP can be used as reporter gene to screen YCD expression vector. YCD / 5_FC suicide gene system can be further used in gene therapy of tumor