【摘要】 目的:鉴定RINm5F细胞中环加氧酶-2(COX-2)启动子区-649/-638转录抑制元件的关键位点及细胞特异性。方法:在RINm5F细胞中,利用荧光素酶报告载体,检测特定位点(-649/-646、-645/-642、-641/-638)突变后启动子活性的改变;通过电泳迁移率转变实验,观察上述位点突变后与细胞核蛋白结合能力的改变。在另外两种细胞系(INS-1、CHO细胞)中,利用荧光素酶报告载体,检测野生型和突变型(-649/-638突变)COX-2的启动子活性。结果:RINm5F中,抑制元件位点-649/-646、-645/-642突变使启动子活性显著上升,与核蛋白结合能力降低。INS-1、CHO细胞中,野生型和突变型启动子活性无显著差异。结论:转录抑制元件的关键位点为-649/-642,此元件具有细胞特异性,可为中医药干预COX-2异常疾病提供明确的作用靶标。更多还原

【Abstract】 Objective:To explore the key nucleotides in the repressor element-649/-638 of COX-2 promoter and its cell specificity.Methods:The luciferase activity of COX-2 promoter was investigated in the pancreatic β-cell line RINm5F using a series of mutant constructs(-649/-646 or-645/-642 or-641/-638 mutant) in order to identify the key nucleotides.The ability of nuclear proteins binding to mutant repressor element was then determined by an electrophoretic mobility shift assay.The luciferase activity of COX-2 wild-type and-649/-638 mutant promoter was investigated in other two cell lines(INS-1,CHO) to clarify whether the repressor element was cell-specific.Results:Mutation of-649/-646 and-645/-642 but not-641/-638 in the COX-2 promoter significantly up-regulated the transcriptional activity and down-regulated the binding activity to nuclear proteins in RINm5F cells.Wild-type and mutant COX-2 promoter activity showed no significant differences in other two cells.Conclusion:The key nucleotides in the repressor element locates between nucleotides-649/-642 of COX-2 promoter.The repressor element is cell-specific.更多还原