Tomita N, Morishita R, Tomita S, Yamamoto K, Aoki M, Matsushita H, Hayashi S, Higaki J, Ogihara T
Department of Geriatric Medicine, Osaka University Medical School, Suita, Japan.
J Hypertens. 1998 Jul;16(7):993-1000. doi: 10.1097/00004872-199816070-00013.
Several cytokines and adhesion molecules released from endothelium play an important role in inflammation, immune responses, and probably atherogenesis.
To determine whether the transcription factor nuclear factor-kappaB mediated expression of these genes involved in the inflammatory response of endothelial cells to tumor necrosis factor-alpha, by using transcription factor decoy oligodeoxynucleotides.
We first transfected fluorescein isothiocyanate (FITC)-labeled double-stranded oligodeoxynucleotides into endothelial cells by a cationic liposome-mediated method of gene transfer. We then confirmed that the decoy oligodeoxynucleotides could block binding of nuclear factor-kappaB to its specific cis element effectively. In addition, we transfected the reporter gene chloramphenicol acetyltransferase driven by three repeated nuclear factor-kappaB binding sequences in the promoter and enhancer region.
FITC-labeled oligodeoxynucleotides were detected in the nuclei of approximately 70% of the total cells. Tumor necrosis factor--stimulated expression of chloramphenicol acetyltransferase was partially inhibited by transfection of nuclear factor-kappaB decoy oligodeoxynucleotides, but not by transfection of scrambled oligodeoxynucleotides. Also nuclear factor-kappaB decoy oligodeoxynucleotides but not scrambled oligodeoxynucleotides inhibited tumor necrosis factor-induced expression of interleukin-6 and intracellular adhesion molecule-1 both at the messenger RNA and at protein level (assessed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay).
Our results demonstrate that nuclear factor-kappaB decoy oligodeoxynucleotides transfected by cationic liposome method inhibited tumor necrosis factor--induced expression of interleukin-6 and intracellular adhesion molecule-1 in endothelial cells.
内皮细胞释放的多种细胞因子和黏附分子在炎症、免疫反应以及可能的动脉粥样硬化形成过程中发挥重要作用。
通过使用转录因子诱骗寡脱氧核苷酸，确定转录因子核因子-κB 是否介导了参与内皮细胞对肿瘤坏死因子-α 炎症反应的这些基因的表达。
我们首先通过阳离子脂质体介导的基因转移方法将异硫氰酸荧光素（FITC）标记的双链寡脱氧核苷酸转染至内皮细胞。然后我们证实诱骗寡脱氧核苷酸能够有效阻断核因子-κB 与其特异性顺式元件的结合。此外，我们转染了由启动子和增强子区域中三个重复的核因子-κB 结合序列驱动的报告基因氯霉素乙酰转移酶。
在约 70% 的总细胞的细胞核中检测到 FITC 标记的寡脱氧核苷酸。转染核因子-κB 诱骗寡脱氧核苷酸可部分抑制肿瘤坏死因子刺激的氯霉素乙酰转移酶表达，但转染随机序列寡脱氧核苷酸则无此作用。同样，核因子-κB 诱骗寡脱氧核苷酸而非随机序列寡脱氧核苷酸在信使核糖核酸和蛋白质水平（通过逆转录-聚合酶链反应和酶联免疫吸附测定评估）均抑制了肿瘤坏死因子诱导的白细胞介素-6 和细胞间黏附分子-1 的表达。
我们的结果表明，通过阳离子脂质体方法转染的核因子-κB 诱骗寡脱氧核苷酸抑制了内皮细胞中肿瘤坏死因子诱导的白细胞介素-6 和细胞间黏附分子-1 的表达。
