【摘要】 目的本文旨在对NGAL基因启动子区及其附近是否存在TPA反应元件进行研究。方法(1)采用PCR法从食管癌细胞中克隆NGAL基因5′侧翼区416～+84片段,然后分别插入质粒pGLB和pGLE,构建pGLB416和pGLE416荧光素酶报告基因表达载体;(2)将pGLB416和pGLE416分别同pRLTK共转染食管癌细胞EC109;(3)用TPA刺激转染的EC109,检测细胞相对荧光素酶活力,通过相对荧光素酶活力变化判定NGAL基因5′侧翼416～+84区是否存在TPA反应元件,同时进行生物信息学分析。结果无论是pGLB416还是pGLE416,与pRLTK共转染食管癌细胞EC109后,用TPA刺激,与其相应的空载体相比,转染细胞荧光素酶活力均极显著升高(t检验,P<0.01),约分别升高46.82和46.41倍;而TPA刺激组与没有TPA刺激组相比,pGLB416和pGLE416转染EC109细胞荧光素酶活力分别显著升高了5.17倍和7.37倍(t检验,P<0.01)。生物信息学分析显示,NGAL基因5′侧翼416～+84区段至少存在4个潜在的TPA反应元件。结论食管癌细胞NGAL基因5′侧翼416～+84区段存在着TPA反应元件。更多还原

【Abstract】 Objective The TPA response elements in NGAL promoter region and the sequences surrounding its promoter region will be determined. Methods The fragment -416~+84 of 5′ flanking region of NGAL gene was amplified from esophageal cancer cells by PCR and then cloned into pGL3-Basic (pGLB) and pGL3-Enhancer vector (pGLE), respectively. Then the constructed recombinant expression plasmid pGLB-416 or pGLE-416 was adopted to cotransfect with pRL-TK into EC109 esophageal cancer cells stimulated with TPA (5ng/ml) or not. Relative luciferase activity of whole cell extracts from cells was measured with Dual Luciferase Report Gene Assay System (DLR) to verify whether there are TPA response elements in the region and the potential positions were analyzed by bioinformatics. Results Relative luciferase activity of either pGLB-416 or pGLE-416 cotransfected with pRL-TK into EC109 cells following stimulation with TPA increased sharply (t test, P< 0.01) by an average of 46.82 or 46.41-fold, compared with controls (empty vector pGLB or pGLE ), and by an average of 5.17 or 7.37-fold than that of unstimulated control. Analysis by bioinformatics showed that there are at least 4 putative TPA responsive elements in -416~+84 position of 5′ flanking region of NGAL gene. Conclusion The TPA response elements of NGAL gene located on -416~+84 position of its 5′ flanking region in the esophageal cancer cells.更多还原