NF-κB decoy对人脐静脉内皮细胞组织因子表达及凝血因子Ⅶ激活抑制作用研究

Study of the inhibitory effect of NF-κB decoy on tissue factor gene expression and FⅦ activation in cultured human umblical vein endothelial cells

目的 用decoy策略调控核因子κB(NF κB)调节组织因子 (TF)表达及相应的因子Ⅶ(FⅦ )活化 ,探讨冠心病新的防治方法。方法 用流式细胞术检测NF κBdecoy对人脐静脉内皮细胞(HUVEC)的转染效率 ,用凝胶迁移率改变竞争实验证实NF κBdecoy的作用机制 ,用RT PCR检测组织因子 (TF)mRNA ,流式细胞术检测HUVEC表面TF抗原 ,重组组织因子一期凝血法检测与HUVEC共孵育的血浆中活化因子Ⅶ (FⅦa)活性。结果 decoy能成功地转染入HUVEC内 ,能与TF启动子的κB位点顺式作用元件竞争性结合转录因子NF κB ,明显抑制肿瘤坏死因子α(TNFα)刺激的HUVECTFmRNATF抗原表达及相应的FⅦa活性。结论 NF κBdecoy能通过抑制TF表达而抑制FⅦ的激活 ,为冠心病的防治提供新的思路。

Objective To investigate the inhibitory effect of NF κB decoy on tissue factor(TF) expression and FⅦ activation in cultured human umbilical vein endothelial cells(HUVEC), and to explore new methods for prevention and treatment of coronary heart disease. Methods NF κB decoy transfection efficiency was detected by flow cytometry, NF κB decoy's mechanism was analyzed by electrophoretic mobility shift assay (EMSA),TF mRNA was detected by RT PCR, TF antigen expression on the surface of HUVEC by flow cytometry, FⅦa level in plasma incubated with HUVEC stimulated by TNF α by rsTF one stage clotting method. Results NF κB decoy could be successfully transfected into HUVEC. It could compete with the endogenous κB cis sequence element in the regulatory regions of TF promoter to bind transcriptional factor NF κB. It could also significantly inhibit the TF mRNA ,TF antigen expression on the cell surface and TF function leading to activation of FⅦ. Conclusion NF κB decoy could inhibit TF gene expression and FⅦ activation in cultured HUVEC and might be a potential new strategy for prevention and treatment of coronary heart disease.