急性肺损伤（ALI）的炎症失衡往往伴随氧化应激损伤，对炎症反应及继发的氧化应激进行调控是减轻 ALI 的关键。前期研究中，我们以TNF-α刺激人II型肺泡上皮细胞A549，观察到炎症反应、氧化应激及细胞凋亡率均上调，同时伴随NF-κB核转位增强，后者与NOX1基因启动子结合，上调其表达。预实验则提示沉默Nrf2基因后，NOX1表达增加，氧化应激失衡加重。既往研究认为，转录因子Nrf2是重要的抗氧化应激调节因子，与急性肺损伤密切相关，但其对NOX家族的调控机制尚不明确。我们推测，Nrf2是调控NOX家族生成ROS的重要节点。鉴此，本课题通过RNA干扰、载体构建、基因突变、EMSA与染色质免疫共沉淀等方法，探讨ALI炎症失衡继发氧化应激过程中Nrf2对NOX家族的调控作用，为将Nrf2作为ALI 基因治疗靶点提供理论与与实践基础。

Imbalance of inflammation is always accomanied with injuries of oxidative stress in acute lung injury (ALI) and the regulation of oxidative strss following inflammation is the key. Our previous study showed that TNF-a activated NF-kB,which bound to NOX1 promoter and increased the NOX1 expression in A549.Furthermore, we found that Nrf2 silencing leaded to the increased NOX1 expression and excessive oxidative stress. It is known that Nrf2 is an important anti-oxidative agent in the process of ALI, but whether regulates the NOX family is unknown. We hypothesize that Nrf2 is a key point in the process of NOX family generating ROS. To achieve the potential valve of Nrf2 in ALI, we are going to explore the effect of Nrf2 in A549 treated with TNF-a. The composite methords,including RNA interference, vector construction, nucleotide mutation, electrophoretic mobility shift assay and CHIPs are to be key steps in our study. We will check several valuable profiles such as Nrf2 expressing, gene transcription regulated by Nrf2. Promisingly, we can elucidate the modulation nechanism of Nrf2, which may be affected by its gene polymorphism between inflammation and oxidative stress in ALI.

急性肺损伤（ALI）的炎症失衡往往伴随氧化应激损伤，对炎症反应及继发的氧化应激进行调控是减轻 ALI 的关键。前期研究中，我们以TNF-α刺激人II型肺泡上皮细胞A549，观察到炎症反应、氧化应激及细胞凋亡率均上调，同时伴随NF-κB核转位增强，后者与NOX1基因启动子结合，上调其表达。预实验则提示沉默Nrf2基因后，NOX1表达增加，氧化应激失衡加重。既往研究认为，转录因子Nrf2是重要的抗氧化应激调节因子，与急性肺损伤密切相关，但其对NOX家族的调控机制尚不明确。本课题组先后成功构建Nrf2 siRNA 及Nrf2 高表达载体，转染入A549 细胞并给予TNF-α刺激，分别在Nrf2低表达与高表达的状态下检测细胞内炎症反应、氧化应激程度、细胞凋亡水平。本研究结果表明：预沉默Nrf2基因，TNF-α诱导的IL-2、IL-6、IL-8等炎症因子水平升高，氧化产物MDA浓度增高，抗氧化物SOD耗损增加，炎症反应放大、氧化应激失衡加重，细胞凋亡率增加( P＜0. 05)。预转染Nrf2高表达载体，TNF-α诱导的IL-2、IL-6、IL-8等炎症因子水平降低，氧化产物MDA浓度下降，抗氧化物SOD耗损减少，炎症反应水平和氧化应激程度减弱，细胞凋亡率降低( P＜0. 05)。结果提示，提高nrf2表达量可降低TNF-α诱导的炎症和氧化损伤，对结构细胞起到保护作用。在此基础上，本课题组应用生物信息学软件分析NOX1 基因启动子区上Nrf2的结合位点，结果提示NOX1近端启动子区约2000bp范围内可预测到2个nrf2结合元件，元件1称为NOX1/nrf2-1，位于-1980~ -1970，序列为5＇- ggctaaggcat - 3＇；元件2称为NOX1/nrf2-2：位于-1199~-1189，序列为5＇- attacacag ca - 3＇。以上述位点的序列为模板合成探针，经EMSA实验检测，元件2（NOX1/nrf2-2）可与核蛋白中nrf2结合。这预示Nrf2可能通过与NOX1 基因启动子区上游-1199~-1189bp的位点结合，调控其表达。本课题目前成果提示Nrf2是调控NOX1基因表达、氧化应激程度的重要节点，上述结果为将Nrf2作为ALI 基因治疗靶点提供基础研究资料，具有重要科学意义。