摘要
目的构建急性肺损伤肺泡Ⅱ型上皮A549细胞炎症模型,通过沉默NF-κB基因,观察NOX1基因表达水平及氧化应激指标变化,探讨NOX1基因与NF-κB/p65的相关性。方法运用RNA干扰技术沉默NF-κB/p65基因,以TNF-α(10 ng/ml)刺激肺泡Ⅱ型上皮细胞(A549),采用RT-PCR及Western blot检测NOX1基因表达水平,DCFH-DA探针法检测细胞内活性氧水平,比色法检测细胞的总抗氧化能力、总谷胱甘肽、总超氧化物歧化酶活力和丙二醛的浓度。结果采用TNF-α刺激肺泡Ⅱ型上皮A549细胞可以分别在基因和蛋白水平上调NOX1基因表达;同时,增加细胞丙二醛和活性氧浓度,降低总抗氧化能力、总谷胱甘肽及超氧化物歧化酶浓度,氧化应激程度放大(P<0.05)。预转染NF-κB/p65 siRNA,可下调NOX1基因的表达,减少细胞丙二醛、活性氧生成,提高总抗氧化能力、总谷胱甘肽及超氧化物歧化酶浓度,氧化应激程度降低(P<0.05)。结论沉默NF-κB/p65基因,可有效下调TNF-α诱导A549细胞氧化应激程度及NOX1基因表达水平,提示NF-κB/p65可能参与调控TNF-α诱导的NOX1基因表达。
This study was performed to establish an in vitro inflammatory cell culture model of acute lunginjury, and observe the expression of NOX1 gene and oxidative stress induced by TNF-α, and to investigate theeffects of NF-κB/p65 on NOX1 by silencing NF-κB/p65 gene. A549 cells were treated with RNAi to down-regulatethe expression of NF-κB, then treated with TNF-α（10 ng/ml）. RT-PCR and Western blotting were performed toanalyze the expression of NOX1 gene. The concentration of ROS was detected by DCFH-DA probe assay, and thecontents of MDA, total antioxidant capacity, SOD and GSH were measured by colorimetric method. Data showed thatthe transcription and translation of NF-κB/p65 were significantly up-regulated in A549 cells stimulated by TNF-α（P〈0.05）. The levels of ROS and MDA were improved, total antioxidant capacity, SOD and GSH were decreasedmarkedly（P〈0.05）. NF-κB/p65 RNAi could down-regulate the expression of NOX1 gene and the oxidative stressinjury, reduce the levels of ROS and MDA, meanwhile, total antioxidant capacity, SOD and GSH were increaseddramatically（P〈0.05）. In conclusions, NF-κB/p65 silencing could decrease the expression of NOX1 geneand reduce oxidative-stress stimulated by TNF-α,suggesting that NF-κB/p65 may play an essential rolein the expression of NOX1 gene.
作者
吴炜景
张家敏
曾奕明
李理
黄文杰
WU Weijing ZHANG Jiamin ZENG Yiming LI Li HUANG Wenjie(Department of Respiratory and Critical Care Medicine, Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, China Department of Respiratory Medicine, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2017年第11期943-948,共6页
Immunological Journal
基金
国家自然科学基金(81541002)
福建省自然科学基金(2015J0102)
福建省卫生厅青年科研课题(2013-2-51)
福建省中青年教师教育科研项目(JB13085)
泉州市科技项目(Z[2013]0206)
