AFP启动子对绿色荧光蛋白在人肝癌细胞中表达的影响  

Impact of AFP Promoter on the Expressions of GFP in Tumor Cells

作　　者：茆灿泉[1] 黄常志[1] 杨树德[1] 赵玫[1] 范云霞[1] 徐枫[1] 杜菲[1] 

机构地区：[1]卫生部北京医院

出　　处：《中国医学科学院学报》2001年第5期495-498,共4页Acta Academiae Medicinae Sinicae

摘　　要：目的研究AFP5′端3.1kb启动子序列对GFP在人肝癌细胞中表达的影响。方法首先采用重组DNA技术,构建了由AFP5′端前导序列AFP启动子+AFPEI增强子,3.1kb调控的绿色荧光蛋白GFP报告基因哺乳动物表达载体pcDNA3-GFP-AFP-w,然后将pcDNA3-GFP-AFP-w、pcDNA3-GFP及空载(pcDNA3-neo)经lipofectin分别转染Bel7402、Hela二种肿瘤细胞,G418抗性筛选得到含各不同质粒的稳定阳性细胞克隆,Westernblotting和密度积分扫描检测GFP基因的表达。结果转染pcDNA3-GFP-AFP-w细胞的GFP表达量在Bel7402中明显高于Hela细胞,在Hela细胞中GFP几乎不表达,但均低于相应转染pcDNA3-GFP的细胞,呈现一定的专一性。结论AFP5′端3.1kb启动子序列调控的GFP报告基因载体pcDNA3-GFP-AFP-w对Bel7402肝癌细胞呈现一定的细胞专一性。Objective To study the impact of AFP5'flanking promoter (enhancer)on the expression of GFP in hepatocarcinoma cell.Methods Green Fluorescent Protein (GFP)reporter gene expression plasmid pcDNA 3 -GFP-AFP-w under the direction of AFP5' flanking promoter(enhancer)was c onstructed by rec ombinant DNA technology and confirmed by restriction analyses .pcDNA 3 -GFP-AFP-w?pcDNA 3 -GFP and pcDNA 3 were transfected into Hela and Bel7402cells by lipofectin and selected b y G418respec-tively,after amplification of the positive cell clones,express ion of GFP was detected by Western blo tt-ing and quantitatively analysised by G EL Doc2000digital image systems.Results The expression of GFP was lower in Bel-GFP-AFP-w than in Bel-GFP but was significantly higher than in Hela-GFP-AFP -w.Conclusion GFP reporter gene plasmid pcDNA 3 -GFP-AFP-w under the direction of the3.1kb AFP5'flanking promoter (enha ncer)can be expressed in HCC Bel7402cell definitely and specifi-cally.

关 键 词：报告基因 GFP AFP启动子 转染 绿色荧光蛋白 肝癌 肝细胞癌 

分 类 号：R735.7[医药卫生—肿瘤] R730.43[医药卫生—临床医学]