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目的:构建核因子-κB(NF-κB)p65特异性小干涉核糖核酸(small interference RNA,siRNA)重组腺病毒载体。方法:设计特异性NF-κBp65siRNA的靶序列对应的双链DNA,插入腺病毒穿梭质粒,然后跟腺病毒骨架质粒共转染HEK-293A细胞,穿梭质粒和骨架质粒在细胞内整合包装重组腺病毒并用β-Gal染色鉴定。重组腺病毒扩增、纯化、滴度测定后,感染培养的大鼠关节软骨细胞,应用RT-PCR在mRNA水平观察NF-κBp65的表达。结果:β-Gal染色显示重组腺病毒载体构建成功,RT-PCR结果显示重组腺病毒有效抑制了目的基因NF-κBp65的表达。结论:特异性NF-κBp65 siRNA的重组腺病载体可以抑制目的基因的表达,可以用于体内抑制骨关节炎的实验研究。
Objective: To construct a recombinant adenovirus vector of nuclear factor-κB (p65) small interfering RNA (siRNA). METHODS: The double-stranded DNA corresponding to the target sequence of specific NF-κBp65siRNA was designed and inserted into the adenovirus shuttle plasmid. The recombinant plasmid was co-transfected with the adenovirus backbone plasmid into HEK-293A cells. The shuttle plasmid and the backbone plasmid were integrated into the recombinant adenovirus And identified by β-Gal staining. The recombinant adenovirus was amplified, purified and titre assayed. The cultured rat articular chondrocytes were infected and the expression of NF-κBp65 was detected by RT-PCR. Results: The results of β-Gal staining showed that the recombinant adenovirus vector was successfully constructed. The results of RT-PCR showed that the recombinant adenovirus effectively inhibited the expression of NF-κBp65. Conclusion: The recombinant adenovirus vector with specific NF-κBp65 siRNA can inhibit the expression of the target gene and can be used in vivo to inhibit osteoarthritis.