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[目的]探索RNA干扰(RNA interference RNAi)技术在抑制人支气管上皮(16HBE)NF-κBp65表达的抑制效应,为后续研究NF-κBp65在环境化学污染物诱导DNA损伤修复中的作用提供实验模型。[方法]设计针对NF-κBp65的siRNA,构建NF-κBp65-shRNA的表达载体,采用阳离子脂载体Lipofectamine2000TM和siPORTTMXP-1两种转染系统比较其抑制率,选择抑制效果较高的转染系统,优化转染条件,从而达到最优的NF-κBp65的抑制模型。[结果]阳离子脂载体Lipofectamine2000TM和siPORTTMXP-1两种转染系统进行比较,siPORTTMXP-1转染系统NF-κBp65的抑制效率更高,约为40%。优化质粒的浓度和转染时间,荧光定量PCR结果质粒浓度为0.4μg,转染48h时NF-κBp65的mRNA抑制率较高,为62.9%。Westernblot检测蛋白质表达水平降低约为60.2%。[结论]RNAi技术可以有效地抑制16HBE细胞中的NF-κBp65表达,该模型的建立有利于后续实验的进行。
[Objective] To explore the inhibitory effect of RNA interference RNAi (RNAi) technology on the inhibition of NF-κBp65 expression in human bronchial epithelial cells (16HBE), and to provide an experimental model for the subsequent study on the role of NF-κBp65 in DNA damage repair induced by environmental chemical pollutants. [Methods] siRNA targeting NF-κBp65 was designed and constructed. The expression vector of NF-κBp65-shRNA was constructed. The antitumor effects of siRNA targeting Lipofectamine2000TM and siPORTTMXP-1 were compared, and the transfection system with high inhibitory effect was selected. Optimize the transfection conditions, so as to achieve the optimal NF-κBp65 inhibition model. [Result] Compared with the two transfection systems of lipofectamine2000TM and siPORTTMXP-1, the inhibitory efficiency of siPORTTMXP-1 transfection system NF-κBp65 was higher, about 40%. The plasmid concentration and transfection time were optimized. The plasmid concentration of fluorescent quantitative PCR was 0.4μg, and the mRNA inhibition rate of NF-κBp65 was 62.9% at 48h after transfection. Westernblot detection of protein expression decreased by about 60.2%. [Conclusion] The RNAi technique can effectively inhibit the expression of NF-κBp65 in 16HBE cells. The establishment of this model is conducive to the follow-up experiments.