NOX1基因荧光素酶报告基因系统的建立

目的：对炎症细胞因子TNF-α及IFN-γ刺激下，人NOX1基因的表达调控进行初步分析。方法：将NOX1基因5′-端上游序列连接到无启动子的PGL3-BASIC质粒，构建了PGL3-BASIC／NOX1报告质粒。PGL3-BASIC／NOX1质粒转染A549细胞，用TNF-α、IFN-γ刺激12h，双荧光素酶报告基因系统检测基因表达情况。结果：克隆的NOX1片段具有较强的启动子活性，在TNF-α和IFN-γ共同刺激下，转染报告基因的A549细胞萤光素酶活性与对照相比有明显的增高（约4．3倍）。分析显示NOX1基因5′端上游序列片段含有NF-KB结合位点，提示细胞因子刺激的荧光素酶表达增强可能与NF-KB位点激活相关。结论：NOX1基因表达水平明显受到炎症细胞因子的调控，提示该基因可能参与机体免疫防御（特别是上皮细胞免疫防御），值得进行深入研究。

Construction of Dual-Luciferase reporter plasmid assay system for human NOX1 gene

Objective： The aim of this study was to investigate the expression and regulation of NOX1 gene in epithelial cells. Methods. 2096 bp of the human NOX1 promoter was amplied with PCR The amplication product was subcloned into the promoterless pGL3-basic rey luciferase vector to generate reporter plasmid pGL3-NOX1. The reporter plasmid was transfected into A549 cells and cells were treated with proinflammation cytokines TNF-α and IFN-γ. The pGL3-NOX reporter plasmid activity was measured by the Dual Luciferase assay system. Results： The expression of pGL3-NOX1 reporter vector in A549 cells was up-regulated significantly by proinflammation cytokines TNF-α and IFN-γ （4. 3 fold increase）. Conclusions： This study shows that proinflammation cyto- kines stimulate NOX1 expression in epithelial cells and NOX1 may play a role in the mucosal inflammatory and immune response.