核因子-κBp65特异性小干涉核糖核酸重组腺病毒的抑制作用

The effect of recombination adenovirus expressing nuclear factor-κBp65-specific small interference RNA in vivo

目的 在大鼠关节内观察核因子-κB（NF-κB）p65特异性小干涉核糖核酸（siRNA）重组腺病毒对靶基因的抑制作用.方法 在SD大鼠膝关节内,筛选合适的病毒剂量.利用β-gal染色鉴定重组腺病毒感染组织的特异性,应用凝胶电泳迁移率分析（EMSA）测定重组腺病毒对NF-κB转录活性的影响,应用Western blot测定重组腺病毒对软骨和滑膜细胞内NF-κB65的抑制作用.结果 重组腺病毒在大鼠膝关节内的最佳剂量为1.6×108 pfu/ml,可以感染关节内软骨和滑膜组织,并在软骨和滑膜细胞内减低NF-κB转录活性,抑制目的 基因的表达.结论 特异性NF-κBp65siRNA的重组腺病毒载体可以在关节软骨和滑膜细胞内抑制目的 基因的表达.

Objective To observe the inhibitory effect of the recombinant adenovirus expressing nuclear factor-κB （NF-κB）p65-specific small interference RNA （siRNA） in knee joints of rats. Methods The recombinant adenovirus was injected into knee joints of SD rats to optimize the dose of the recombinant adenovirus. The staining of β-gal was used to determine the tissue specificity of the recombinant adenovirus, the effect of recombinant adenovirus on activation of NF-κB was measured with electrophoretic mobility shift assay （ EMSA）, and Western blotting was used to measure the effect of the recombinant adenovirus on the expression of NF-κB in chondrocytes and synovial cells. Results The optimal dose of the recombinant adenovirous in SD rat joints was 1.6 × 108 pfu/ml. Staining of β-gal indicated that the recombinant adenovirus could be transfected into the cartilage and synovium. EMSA and Western blotting revealed that the recombinant adenovirus could be expressed in chondrocytes and synovial cells, reduce the activation of NF-κB and inhibit the expression of NF-κBp65. Conclusion The recombinant adenovirus expressing NF-κBp65-specific siRNA can reduce the activation of NF-κB, and inhibit the expression of NF-κBp65 in knee joints of rats.