硫代磷酸酯寡核苷酸的合成及其对内皮细胞组织因子(Tissue factor,TF)基因表达及TF促凝活性的作用

为合成与 TF基因启动子区切应力反应元件 ( Shear stress responsive element,SSRE)形成三链 DNA的硫代磷酸酯寡核苷酸 ( Triple helix- forming phosphorothioate oligodeoxynucleotides,TFO- ps) ,探讨其对内皮细胞TF基因表达及 TF促凝活性的影响。我们设计反向 TFO ( T2 1GTa)序列 ,采用固相亚磷酰胺三酯固相法合成TFO。在 TFO的 3′末端进行硫代磷酸酯修饰。应用电泳迁移分析 ( Electrophoretic mobility shift assays,EMSA)观察寡核苷酸和硫代脱氧寡核苷酸的亲和性。在 ECV3 0 4内皮细胞株观察 γ- 32 P标记硫代磷酸酯 TFO的细胞摄取及其对 TF基因表达的影响。结果显示 ,TFO- ps( T2 1Gta- ps)与靶序列能形成三链 DNA,其 Kd值为 3 .6× 10 - 1 0 M。在内皮细胞株 ECV3 0 4细胞 ,TFO- ps( T2 1GTa- ps)的细胞吸收率为 11.65 % ,主要分布在核沉淀 ,约占吸收总量的77.2 5 % ,显著降低内皮细胞 TF基因 m RNA表达及 TF蛋白合成的平均光密度 ( AOD)值 ,显著降低 TF的促凝活性。以上结果表明 ,TFO- ps( T2 1GTa- ps)具有较好的抗凝活性 ,其机制与抑制 TF基因表达有关

Synthesis of a Triple Helix-forming Phosphorothioate Oligodeoxynucleotides and Its Effects on Coagulation Activity of Tissue Factor(TF)and TF Gene Expression in Endothelial Cells

This study sought to synthesize a triple helix-forming phosphorothioate oligodeoxynucleotides (TFO-ps) and assess its effects on coagulation activity of the tissue factor(TF) and TF gene expression in endothelial cells. Experiment antiparallel oligodeoxynucleotides T21GTa sequence was designed and synthesized by phosphoramidite method and decorated with all-PS linkage. The affinity of TFO and TFO-ps was determined by electrophoretic mobility shift assays(EMSA). Cellular uptake of 32]P-labeled TFO-ps and the effect of TFO-ps on TF gene expression and coagulation activity of TF were measured in endothelial cell strain ECV304. The results showed that TFO-ps (T21GTa-ps) formed a triplex binding in antiparallel orientation to the puring-rich target strand-SSRE, with Kd value of 3.6 x 10(-10) M. The uptake rate of TFO-ps (T21GTa-ps) in ECV304 was about 11.65%. This compound mainly localized within the nucleus sediment(77.25%), significantly reduced the average OD of the mRNA expression and protein synthesis of TF gene, and obviously decreased the coagulation activity of TF. In conclusion, TFO-ps (T21GTa-ps) shows obvious anti-coagulation activity and its mechanism involves the inhibition of TF gene expression.