摘要
目的:构建人CD133基因启动子启动加强型绿色荧光蛋白(eGFP)基因表达载体,观察在喉癌Hep-2细胞系中人CD133基因启动子启动eGFP基因表达能力,为应用CD133基因启动子调控靶向肿瘤干细胞(CSCs)基因治疗奠定实验基础。方法:采用PCR方法从人外周血基因组中克隆人CD133基因启动子,通过基因重组技术将人CD133基因启动子克隆入慢病毒基因转移载体pWPXLd-eGFP中,构建人CD133基因启动子启动eGFP基因表达载体,PCR以及酶切鉴定构建载体的正确性。应用脂质体介导该载体转染入Hep-2细胞中,细胞分为空白对照组(未转染质粒)、阳性对照组(转染pWPXLd)和实验组(转染pWPXLd-eGFP-CD133)。应用荧光显微镜观察各组eGFP基因在喉癌Hep-2细胞内的表达。结果:PCR法获得了约1 810bp大小的CD133基因启动子片段;酶切与PCR鉴定,成功构建了人CD133基因启动子启动eGFP基因表达载体。荧光显微镜下,阳性对照组和实验组转染的Hep-2细胞株有eGFP的表达,而空白组对照组的Hep-2细胞内无eGFP的表达。结论:构建的人CD133基因启动子启动eGFP基因表达载体可以调控eGFP基因在喉癌Hep-2细胞内的表达。
Objective To construct the gene expression vector of enhanced green fluorescent protein（eGFP）regulated by human CD133 gene promoter and to observe the expression ability of the human CD133 gene promoter in laryngocarcinoma Hep-2 cell line,and to provide experimental basis for the target therapy of tumor stem cells（CSCs） regulated by human CD133 gene promoter.Methods The CD133 gene promoter was cloned from the genomic DNAs of human peripheral blood by PCR method and cloned into lentivirus pWPXLd-eGFP by using gene recombination technology to construct eGFP gene expression vector induced by human CD133 gene promoter.The recombined vector was identified by PCR and enzyme digestion.The recombined vector was transfected into Hep-2 cells induced by liposome.The cells were divided into blank control group（non-transfected with plasmid）,positive control group（transfected with pWPXLd）,and experiment group（transfected with pWPXLd-eGFP-of CD133）;and the expression of eGFP gene in Hep-2 cells was observed under fluorescence microscope.Results The specific 1 810 bp CD133 gene promoter fragment was obtained successfully by PCR method.The eGFP gene expression vector regulated by human CD133 promoter was constructed successfully by PCR and enzyme digestion method.The eGFP expressions in Hep-2 cells in positive control group and experiment group were found,but weren＇t in blank control group under fluorescence microscope.Conclusion The eGFP gene expression vector regulated by human CD133 gene promoter can regulate the expression of eGFP gene in laryngocarcinoma Hep-2 cells.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2013年第3期498-502,I0001,共6页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅科研基金资助课题(201201060
201215078)
吉林省卫生厅科研基金资助课题(2011Z048)
