摘要
【目的】构建自噬基因Beclin1启动子报告基因细胞自噬模型,为筛选出以Beclin1为靶标的中药提供新的细胞模型。【方法】采用瞬时转染双荧光素酶报告基因测定法,确定最佳转染浓度、时间等条件,及Beclin1基因启动子转录活性细胞自噬模型的建立和验证,观察血清饥饿、过氧化氢氧化损伤及自噬激活剂雷帕霉素3种自噬刺激因素对Beclin1基因启动子转录活性PC12细胞自噬模型的影响。【结果】血清饥饿诱导12 h,Beclin1基因启动子转录活性是体积分数10%血清组的1.8倍,而血清饥饿诱导18、24 h与诱导12 h比较其转录活性无明显变化;300μmol/L过氧化氢诱导9 h,Beclin1基因启动子转录活性是0μmol/L过氧化氢组的1.3倍;雷帕霉素在浓度0.1、1 nmol/L可上调Beclin1基因启动子转录活性,并呈浓度依赖性,而5 nmol/L及更高浓度的雷帕霉素可下调Beclin1基因启动子转录活性。【结论】成功建立用Beclin1基因启动子报告基因检测Beclin1基因启动子转录活性特异性的细胞发生自噬模型,血清饥饿、过氧化氢、自噬激活剂雷帕霉素3种自噬刺激因素可诱导Beclin1基因启动子的转录活性。
Objective To construct an autophagic cell model with Beclin1 promoter reporter gene for screening Beclin1-targeting Chinese herbs. Methods Transient transfection with dual luciferase for reporter gene method was used to screen the optimal Beclin1 transfection concentration and time, and to verify the autophagic cell model with Beclin1 promoter reporter gene. After the establishment of the model, we observed the effect of autophagy-stimulating factors of serum starvation, hydrogen peroxide injury, rapamycin（ an autophagic activator） on transcriptional activity of beclin1 promoter in autophagic PC12 cell model. Results Beclin1 promoter transcriptional activity after serum starvation for 12 hours was 1.8 times higher than that in 10% serum control group, but did not differ from that after serum starvation for 18 and 24 hours. Beclin1 promoter transcriptional activity after induction with 300 μmol / L hydrogen peroxide for 9 hours was 1.3 times higher than0 μmol / L hydrogen peroxide group. Beclin1 promoter transcriptional activity was up-regulated by Rapamycin at0.1 and 1 nmol / L in concentration-dependant manner, but was down-regulated by 5 nmol / L or more higher concentration of Rapamycin. Conclusion The autophagic cell model has been successfully established, in which beclin1 specific transcriptional activity can be determined by transient transfection with dual luciferase for reporter gene method. Serum starvation, hydrogen peroxide injury and Rapamycin（ an autophagic activator）can induce transcriptional activity of beclin1 promoter.
出处
《广州中医药大学学报》
CAS
2015年第2期325-330,388,共7页
Journal of Guangzhou University of Traditional Chinese Medicine
基金
国家自然科学基金项目(编号:81260530)
广东省自然科学基金项目(编号:S2012010010406)
