针对SP1的圈套寡脱氧核苷酸抑制NIH_3T_3细胞活力和Ⅲα_1胶原基因表达的研究  

A decoy-oligodeoxynucleotide against transcriptional factor SP1 can suppress cell viability and type Ⅲ collagen's gene expression in cultured NIH_3T_3

作　　者：张世新[1] 杨松林[1] 万伟东[2] 史毅[3] 邓辰亮[1] 金由辛[3] 

机构地区：[1]上海交通大学附属第六人民医院整形外科,上海200233 [2]东南大学附属中大医院整形外科 [3]中国科学院上海生命科学研究院生化细胞所

出　　处：《中国美容医学》2008年第2期217-221,共5页Chinese Journal of Aesthetic Medicine

基　　金：国家自然科学基金资助项目(编号:30571929);基金名称:正义寡核苷酸抑制疤痕增殖相关基因转录的实验研究

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。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 DecoyODN 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.

关 键 词：圈套寡脱氧核苷酸(Decoy—ODN) 转录因子SP1 细胞活力 Ⅲα1胶原 成纤维细胞 

分 类 号：Q813[生物学—生物工程]