作　　者： ; ; ; ; ;

机构地区： 广州医学院

出　　处： 《现代生物医学进展》 2011年第20期3853-3856,共4页

摘　　要： 目的:研究β-萘黄酮对荧光素酶活性的影响。方法:利用人GCLC基因调控序列驱动的GCLC-PGL3-enhancer-Luciferase报道载体(PL45)转染人肺腺癌细胞A549,人肝癌细胞HepG2,人子宫颈癌细胞HeLa,人乳腺癌细胞MCF-7,人肝癌细胞Bel-7402,人支气管上皮细胞16HBE,β-萘黄酮刺激后,双荧光素酶报告基因检测系统分析其对GCLC基因表达的影响。wester blot检测β-NF刺激16HBE细胞后GCLC蛋白水平的变化。β-萘黄酮刺激转染了表达Luciferase的真核表达载体pRC/CMV2-luc+的A549和HepG2细胞后,双荧光素酶报告基因检测系统分析其对Luciferase基因表达的影响。PL45转染A549和HepG2细胞,裂解细胞后用β-NF刺激,双荧光素酶报告基因检测系统分析其对Luciferase基因的影响。结果:在各种细胞中,转染PL45报道载体后,β-NF处理组荧光素酶相对活性值与DMSO对照组相比均明显下降(P<0.01)。wester blot结果显示β-NF处理组GCLC蛋白的表达较DMSO对照组明显升高。在A549和HepG2细胞中,转染pRC/CMV2-luc+载体后,β-NF处理组荧光素酶相对活性值与DMSO对照组相比均明显下降(P<0.01)。PL45转染A549和HepG2细胞,裂解细胞后用β-NF刺激,β-NF处理组荧光素酶相对活性值与DMSO对照组相比均明显下降(P<0.01)。 Objective： To investigate the effect of β-Naphthoflavone on the firefly luciferase activity. Methods： A549, HepG2, HeLa, MCF-7, Bel-7402, 16HBE cells were transfected with GCLC5＇-upstream regulatory sequence driven PGL3-enhancer-Luciferase reporter vector （PIA5）and treated with β-NF. The dual-luciferase reporter assay system was used to analyze the effect of β-Naphthoflavone on the expression of GCLC gene. Western blot was used to detect the change of protein level. A549 and HepG2 cells were transfected with the eukaryotic expression vector pRC/CMV2- luc＋ and treated with β-NF. The dual-luciferase reporter assay system was used to analyze the effect of β-Naphthoflavone on the firefly luciferase activity. A549 and HepG2 cells were transfected with the PIA5 vector, cells were lysed, then treated with β-NF for 25 rain to analyze the effect of β-NF on the firefly lucifemse activity. Results： In all the cells, the relative luciferase activity of β-NF treatment group was significantly lower compared with that in DMSO control group （P〈0.01）. The result of western bot showed that the expression of GCLC were higher inl3-NF treatment group than that in DMSO control group, in A549 and HepG2 cells, after transfected with pRC/CMV2- luc＋ vector, the relative luciferase activity of β-NF treatment group was lower than that in DMSO control group （P〈0.01）.A549 and HepG2 cells were transfected with PL45 vector, then lysed, and the relative luciferase activity in β-NF treatment group was lower than that in DMSO control group （P〈0.01）.Conclusion： β-Naphthoflavone directly inhibits Firefly Luciferase activity.

关 键 词： 萘黄酮 基因转录调控 荧光素酶 报告基因

领　　域： [生物学] [生物学]