【摘要】 目的:研究针对转录因子SP1的圈套寡脱氧核苷酸(Decoy-Oligodeoxynucleotide,Decoy-ODN)对小鼠胚胎成纤维细胞(NIH3T3)的活力和Ⅲα1胶原基因表达的影响,探讨圈套寡脱氧核苷酸用于病理性瘢痕基因治疗的可能性及机理。方法:设计合成针对SP1的特异性哑铃形Decoy-ODN。用阳离子脂质体转染NIH3T3细胞。分别用流式细胞仪检测ODN转染效率,激光共聚焦显微镜观察ODN在细胞中的分布,电泳迁移率变动分析(electrophoretic mobility shift assay,EMSA)验证ODN与SP1的特异性结合。用WST-8检测ODN对细胞活力的影响。用RT-PCR检测ODN对细胞胶原基因表达的抑制作用。结果:分别转染25nM、50nM、100nM、150nM SP1 Decoy-ODN,48h后,细胞活力依次为0.9331±0.0203、0.7479±0.0868、0.577±0.0347、0.4703±0.0147;转染100nMSP1Decoy-ODN可明显抑制Ⅲα1胶原mRNA的表达(P<0.01),抑制效果达60%。结论:Decoy-ODN可以通过拮抗核转录因子SP1的活性而抑制NIH3T3细胞的活力和Ⅲα1胶原基因的表达。更多还原

【Abstract】 Objective To investigate the effect of decoy-oligodeoxynucleotide(Decoy-ODN) against transcription factor SP1 on cell viability and expression of α1 type Ⅲ collagen in mouse embryo fibroblasts(NIH3T3). Methods Dumb-bell Decoy-ODN targeting SP1 was designed and synthesized.SP1 Decoy-ODN was transfected into NIH3T3 cells by cationic liposomes.The transfection efficiency was detected by flow cytometry.The distribution of Decoy-ODN in the cells was investigated by laser scanning confocal fluorescence microscope.The specific binding of Decoy-ODN with SP1 was determined by electrophoretic mobility shift assay(EMSA).The cell viability was examined by WST-8. The effect of decoy-ODN on the collagen’s gene expression was analyzed by RT-PCR. Results Cell viability of NIH3T3 transfected with 25nM、50nM、100nM、150nM SP1 Decoy-ODN was 0.9331±0.0203、0.7479±0.0868、0.577±0.0347、0.4703±0.0147 respectively after 48h;100nM of SP1 Decoy-ODN could effectively inhibit the expression of α1 type Ⅲ collagen(P<0.01), the effect of inhibition was up to 60%. Conclusion SP1 Decoy-ODN could inhibit the cell viability and the expression of α1 type Ⅲ collagen by antagonizing SP1.更多还原