摘要
目的:观察核转录因子(NF)-κB圈套(decoy)对脂多糖(LPS)刺激后人脐静脉内皮细胞NF-κB激活的抑制作用,并就其作用机制进行探讨。方法:获取和培养人脐静脉血管内皮细胞(HUVECs),平均分成4组。NF-κB-圈套组和错配-圈套组分别预先以NF-κB-圈套-Lipofectamine2000复合物和错配-圈套-Lipofectamine2000复合物(对照组)处理,LPS组和对照组分别加入同体积的培养液,观察荧光标记DNA转染细胞情况。用10μg/mlLPS刺激2h激活NF-κB,收集细胞,分别提取细胞核蛋白,检测不同组NF-κB活性以及P65和P50亚基蛋白质含量。结果:转染5h后大于90%细胞核内可观察到绿色荧光。NF-κB-圈套组NF-κBP65亚基活性显著低于错配-圈套组和LPS组,但P65和P50亚基蛋白质含量两组之间无显著差异。结论:NF-κB-圈套能抑制细胞核内NF-κB的DNA结合活性,这一作用可能是NF-κB-圈套转入细胞核后占据转录因子核定位区的结果。
Objective To examined whether the Nuclear factor-kB(NF-κB)-decoy stratagy will inhibit lipopolysaccharide(LPS)-induced NF-κB activation in cultured endothelial cells, and to explore its mechanism. Methods Human umbilical vein endothelial cells (HUVECs) were procured from human umbilical cord veins. HUVECs were incubated with FITC labeled NF-κB or scrambled decoy for 5 hours, and then stimulated the cells with LPS for another hour at a final concentration of 25 μg/ml. Nuclear extract was prepared from HUVECs, and the DNA-binding activity of P65 was compared between groups. At the same time, nucleus P50 and P65 protein levels were determined by western blotting assay. Results Fluorescence was detected in more than 90% of the cells 5 hours after transfection. NF-κB-decoy, but not scrambled decoy, inhibited the NF-κB activation. But the nucleus P50 and P65 levels were not affected in either the NF-κB or scrambled-decoy groups. Conclusions NF-κB-decoy masks the DNA-binding activity of NF-κB, but does not affect transcription factor nucleus translocation.
出处
《外科理论与实践》
2005年第3期248-251,共4页
Journal of Surgery Concepts & Practice
