以分泌型荧光素酶为报告基因的Nrf2报告系统的建立

摘要

Objective: To establish the Nrf2 signaling pathway activation reporting system with secreted Gaussia luciferase ( Gluc) as report gene. Methods: Different numbers ( 1, 2, 4 or 8) of antioxidant-re-sponse elements ( ARE) were inserted into the upper area of mini-ML and mini-CV based as transcribed se-quences by molecular cloning method. The activities of Gluc with different transcribed sequences were com-pared. Alkaline phosphatase driven by CMV promoter was used as a reference gene to constructed plasmids. Then the plasmids was transfect into HaCaT cells and the Gluc or AP activity of HaCaT cells treated by sul-foraphane ( SF) and Tedinki ( BHQ) hydroquinone was detected. Results: The more ARE elements in tran-scribed sequences, the higher the activity of Gluc was. The interference of mini-ML to antioxidant was lower than that of mini-ML. The Gluc activity of HaCaT cells with transcribed sequences of ML-8ARE plasmid was the highest at 6 hours after treated by SF and TBHQ and at 24 hours after radiated by UV. Conclusion: The Nrf2 signaling pathway activation reporting system with secreted Gaussia luciferase ( Gluc) as report gene is successfully constructed, which lays the foundation for the model of UV diseases.%目的:建立以分泌型荧光素酶Gaussia Luciferase(Gluc)为报告基因的Nrf2信号通路的报告系统.方法:以mini-ML和mini-CV为基础转录序列,通过分子克隆方法在其上游分别串联1个、2个、4个和8个抗氧化反应元件(Anti-oxidant response element,ARE),检测不同转录元件下Gluc的活性;同时以CMV启动子驱动的碱性磷酸酶(Alkinane Phosphtase,AP)为内参基因,构建报告质粒.将上述质粒转染到HaCaT细胞,在莱菔硫烷(SF)和特丁基对苯二酚(BHQ)作用的情况下,测定Gluc和AP的活性.结果:转录序列串联的ARE元件越多,Gluc活性越高;以mini-ML为基础转录序列的质粒较mini-CV为基础转录序列的质粒对抗氧化活性的干扰小;以mini-ML为基础转录序列串联8个ARE元件的HaCaT细胞中在SF和BHQ处理6 h、UV辐射后4 h时,Gluc活性最大.结论:本研究成功构建了以Gluc为报告基因的Nrf2报告系统,该系统为UV疾病细胞模型的建立奠定了基础.