重组腺病毒ΔN IκBα的构建及其对A549细胞中NF-κB活性的抑制  

Construction of recombinant ΔN IκBα adenovirus and its suppression of NF-κB activity in A549 cells

作　　者：朱自路[1] 程志祥[1] 熊俊[1] 陈丙莺[1] 

机构地区：[1]南京医科大学生物化学与分子生物学系,江苏南京210029

出　　处：《细胞与分子免疫学杂志》2005年第2期171-174,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：南京医科大学创新基金资助项目(No. 2003 1913)

摘　　要：目的: 探讨ΔNIκBα基因对NF -κB活性的调节作用。方法: 构建去除Ser32和Ser36磷酸化位点的IκBα重组腺病毒Ad- ΔNIκBα。A549细胞分为 3组: 即LPS组、Ad- LacZ+LPS组和Ad -ΔNIκBα+LPS组。LPS组单纯用内毒素(LPS)激活NF -κB; Ad -LacZ+LPS组及Ad ΔNIκBα+LPS组在用LPS前 2d, 分别感染Ad -LacZ和Ad- ΔNIκBα。用West ernblot、电泳迁移率变动分析 (EMSA)和ELISA法分别检测LPS刺激后 5h, 细胞总蛋白中NF κB的活性和培养上清中TNF -α及IL- 6的含量。结果: Ad -ΔNIκBα+LPS组NF -κB的活性, TNF- α和IL -6的含量, 均显著低于LPS组及Ad- LacZ+LPS组。结论: 突变后的IκBα可明显抑制NF- κB活化, 减少TNF- α和IL- 6的释放, 有望成为一种强有力的抗炎治疗制剂。AIM: To explore the regulatory effect of ΔN IκBα gene on the activity of nuclear factor-κB(NF-κB). METHODS: Ser 32-and Ser 36-deleted IκBα gene (ΔN IκBα) was cloned into adenovirus vector, and a replication-defective recombinant ΔN IκBα adenovirus(Ad-ΔN IκBα) was generated. A549 cells were divided into three groups: LPS-stimulated groups, Ad-LacZ+LPS group and Ad-ΔN IκBα+LPS group. Ad-LacZ+LPS group and Ad-ΔN IκBα+LPS group were infected with Ad-LacZ and Ad-ΔN IκBα, respectively, two days before LPS stimulation. The NF-κB activity of A549 cells was detected by Western blot and electrophoretic mobility shift assay (EMSA). TNF-α and IL-6 in the culture supernatant were detecteded by ELISA. RESULTS: The activity of NF-κB and the levels of TNF-α and IL-6 from Ad-ΔN IκBα virus-infected A549 cells were significantly decreased as compared with that of LPS-stimulated group and Ad-LacZ+LPS group. CONCLUSION: The results indicated that ΔN IκBα may inhibit the activivation of NF-κB and reduce the release of TNF-α and IL-6, suggesting the recombinant ΔN IκBα adenovirus may be used for anti-inflammatory therapy.

关 键 词：NF—kB IKB 重组腺病毒 

分 类 号：Q782[生物学—分子生物学]