沉默NF-κB p65基因下调TNF-α诱导的肺泡上皮细胞的炎症反应

NF-κB p65 silencing by RNAi downregulates the inflammation in TNF-α-activated type Ⅱ alveolar epithel cell

目的建立急性肺损伤体外细胞炎症模型,探讨NF-κB p65基因沉默减轻细胞炎症、保护肺结构细胞的可行性。方法以TNF-α(10 ng/ml)刺激肺泡Ⅱ型上皮细胞(A549),运用RNA干扰技术沉默NF-κB p65基因,采用RT-PCR及Westernblotting法检测沉默效率,ELISA法检测细胞培养上清中IL-1β、IL-8、IL-10等炎症因子浓度。结果 TNF-α刺激A549细胞可在基因水平上调NF-κB p65的表达,并增加NF-κB蛋白的核转位,上调细胞培养上清中IL-1β、IL-8、IL-10的浓度;预转染NF-κB p65 siRNA可在基因水平及蛋白水平有效沉默NF-κB p65表达,降低上述各炎症因子浓度(P<0.05)。结论急性肺损伤体外细胞炎症模型构建成功,RNA干扰技术能有效沉默该模型NF-κB p65基因,下调炎症反应水平,保护肺泡上皮细胞,为急性肺损伤免疫调控机制的研究和基因靶向治疗提供实验依据。

Acute lung injury（ALI） is associated with excessive inflammation caused by high TNF-α concentration and over-activity of NF-κB.It has been hypothesized that suitable down-regulation of NF-κB p65 can slow down the ＂waterfall＂ of the inflammation triggered by TNF-α and protect the structure cells of lung in ALI.The present study was performed to investigate the effects of NF-κB p65 silencing by RNAi in type Ⅱ alveolar epithelial cell（A549）.A549 cells were treated with TNF-α（10 ng/ml,24 h） in the absence or presence of NF-κB p65 siRNA.Then RT-PCR and Western blotting were performed to analyze the silence efficiency of RNAi targeting NF-κB p65.The contents of IL-1β,IL-8 and IL-10 in the culture supernatant were measured by ELISA.We found that p65 RNAi significantly decreased the transcription and translation of NF-κB/p65 stimulated by TNF-α（P 0.05）.The levels of IL-1β,IL-8 and IL-10 were lower markedly in the supernatants of A549 cells pre-transfected with NF-κB p65 siRNA（P 0.05）.In conclusion,NF-κB/p65 silencing could down-regulate the over inflammation induced by TNF-α,and NF-κB/p65 RNAi maybe a valuable tool of gene therapy for ALI treatment in the future.