Li Qianning, Ying Dajun, Dai Guangming, Zheng Jian
Biomechanical Laboratory, Department of Anatomy, Third Military Medical University, Chongqing 400038.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2003 Mar;20(1):71-5, 90.
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.
本研究旨在合成一种形成三链螺旋的硫代磷酸寡脱氧核苷酸（TFO-ps），并评估其对组织因子（TF）凝血活性及内皮细胞中TF基因表达的影响。通过亚磷酰胺法设计并合成了反平行寡脱氧核苷酸T21GTa序列，并用全硫代磷酸酯键修饰。采用电泳迁移率变动分析（EMSA）测定TFO和TFO-ps的亲和力。在ECV304内皮细胞株中检测[32]P标记的TFO-ps的细胞摄取情况以及TFO-ps对TF基因表达和TF凝血活性的影响。结果显示，TFO-ps（T21GTa-ps）与富含嘌呤的靶链-SSRE以反平行方向形成三链结合，解离常数（Kd）值为3.6×10（-10）M。TFO-ps（T21GTa-ps）在ECV304中的摄取率约为11.65%。该化合物主要定位于细胞核沉淀中（77.25%），显著降低了TF基因mRNA表达和蛋白质合成的平均光密度（OD），并明显降低了TF的凝血活性。综上所述，TFO-ps（T21GTa-ps）具有明显的抗凝活性，其机制涉及对TF基因表达的抑制。
