核因子-κB圈套对Kupper细胞活化的抑制作用及其机制

Inhibitory effects of NF.κB decoy oligodeoxynucleotides on Kupffer cell activation and mechanism

目的探讨核因子-kB(NF-kB)圈套对大鼠Kupper细胞(KCs)活化的抑制作用及其机制。方法分离大鼠KCs并随机分为3组：正常对照组、内毒素(LPS)刺激组及NF-kB圈套组。后者KCs在LPS刺激前转染NF-kB圈套。除对照组外,两个实验组培养液中加入终浓度为1mg/ L的LPS。于LPS刺激6h后,凝胶电迁移率改变分析法(EMSA)检测NF-kB蛋白结合活性,逆转录-聚合酶链反应(RT-PCR)检测KCs膜表面CD80 mRNA表达,酶联免疫吸附试验(ELISA)检测培养上清肿瘤坏死因子(TNF)-α和白细胞介素(IL)-6表达情况。结果转染NF-kB圈套可以高效抑制KCs激活,EMSA结果显示NF-kB活性仅为LPS组的0．53,RT-PCR结果显示KCs CD80 mRNA表达仅为LPS组的0．46,ELISA结果显示培养上清TNF-α表达量仅为LPS组的0．37,IL-6仅为LPS组的0．60。结论NF-kB圈套可以高效抑制NF-kB的转录活性,降低KCs膜表面共刺激分子和细胞因子的产生,为活体内应用NF-kB圈套提供了可靠的实验依据。

Objective To investigate the inhibitory effects and mechanism of NF-κB decoy oligodeoxynucleotides （ODN） on Kupffer ceUs （KCs） activation. Methods KCs were isolated with in situ coUagenase perfusion method and were randomly divided into three groups： control group, LPS stimulation group （1 mg/L LPS）, and NF-κB decoy group, in which the KCs were transduced with NF-κB decoy ODN （4 μg/1 ×10^5 KCs） prior to LPS stimulation. FoUowing 6 h of LPS stimulus, NF-κB activity was assayed by electrophoretic mobility shift assay （EMSA）, CD80 mRNA expression in KCs was detected by reverse transcription-polyrnerase chain reaction （RT-PCR）, and the production of TNF-α and IL-6 in supernatant was measured by ELISA. Results NF-κB decoy ODN could efficiendy inhibit KCs activation by LPS stimulus. NF-κB activity was significantly decreased to 0.53 fold compared with LPS group. CD80 mRNA expression, TNF-α production and IL-6 level were significantly decreased to 0.46, 0.37 and 0.60 fold, respectively. Conclusion NF-κB decoy ODN can efficiently suppress transcription activity of NF-κB and inhibit costimulatory molecules and cytokines expression by KCs, which afford reliable experimental data for application of NF-κB decoy ODN in vivo.