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目的检测针对组织因子(TF)基因启动子区切应力反应元件(shear stress response elem ent,SSRE)设计的反向硫代形成三链DNA的寡核苷酸(antiparallel phosphoroth ioate trip lex-form ing oligonuc leotide,apsTFO)对大鼠颈总动脉狭窄处内皮细胞TF表达的影响。方法采用硅胶管套扎法建立SD大鼠颈总动脉中段重度狭窄模型。应用原位杂交和免疫组织化学方法结合图像分析系统测定狭窄段内膜TF,Egr-1和Sp1的mRNA表达及蛋白合成。术前0.5 h在尾静脉注射0.5 mg.kg-1针对TF的SSRE结合位点设计合成的GT20-apsTFO,GT20-psTFO,GT21-apsTFO及部分绿色荧光素(FITC)标记的apsTFO,术后4 h检测血管内皮细胞内的TFO,术后6 h检测TFO对TF的抑制效果及对Egr-1和Sp1的影响。结果给予TFO后,在血管内皮细胞核中可见荧光分布。GT20-apsTFO和GT21-apsTFO对TF的mRNA表达和蛋白合成有显著性抑制(P<0.05),且GT20-apsTFO的抑制作用较强,与GT21-apsTFO组相比差异有显著性(P<0.05),GT20-psTFO对TF的mRNA表达和蛋白合成无明显抑制(P>0.05)。GT20-apsTFO,GT20-psTFO和GT21-apsTFO对Egr-1及Sp1的mRNA表达和蛋白合成无抑制作用。结论apsTFO能部分进入血管内皮细胞并在一定程度上抑制TF基因的表达,而不影响Egr-1和Sp1基因的表达。
Objective To detect the effect of antiparallel phosphorothioate trip lex-form ing oligonucotide (FRA) on the design of shear stress response elem ent (SSRE) in the promoter region of tissue factor (TF) , apsTFO) on endothelial cell TF expression in rat common carotid artery stenosis. Methods The stenosis model of middle carotid artery in SD rats was established by silicone tube ligation. In situ hybridization and immunohistochemistry were combined with image analysis system to determine the mRNA and protein synthesis of TF, Egr-1 and Sp1 in the stenosis. The apsTFO labeled with GT20-apsTFO, GT20-psTFO, GT21-apsTFO and FITC were designed by intracerebral injection of 0.5 mg.kg-1 of 0.5 mg.kg-1 for 0.5 h before surgery 0.5 h before surgery. h to detect TFO in vascular endothelial cells. The inhibitory effect of TFO on TF and the effect on Egr-1 and Sp1 were detected 6 hours after operation. Results After giving TFO, fluorescence distribution was observed in the nucleus of endothelial cells. GT20-apsTFO and GT21-apsTFO significantly inhibited TF mRNA expression and protein synthesis (P <0.05), and GT20-apsTFO had a stronger inhibitory effect than GT21-apsTFO group (P <0.05) ), GT20-psTFO had no significant inhibition on mRNA and protein synthesis of TF (P> 0.05). GT20-apsTFO, GT20-psTFO and GT21-apsTFO had no inhibitory effect on the mRNA expression and protein synthesis of Egr-1 and Sp1. Conclusion ApsTFO can partially enter vascular endothelial cells and inhibit the expression of TF gene to a certain extent without affecting the expression of Egr-1 and Sp1 genes.