摘要
目的在炎症氧化应激细胞模型中筛选参与Nox1启动子活化的相关调控蛋白。方法 TNF-α刺激A549细胞建立炎症氧化应激细胞模型,运用DNApull-down技术筛选出Nox1启动子区结合蛋白,再用二维凝胶电泳技术分离pull-down后的结合蛋白,然后在2D凝胶上选取与对照组表达差异大于1.5倍的蛋白质点进行切胶,最后经基质辅助激光解吸电离飞行时间质谱鉴定,筛选出Nox1启动子区差异结合蛋白。结果 2D凝胶上共筛选出1.5倍以上差异蛋白点7个,均为表达上调蛋白,鉴定出GLE1、DDX19A,KRT1、KRT1O四种蛋白质。结论 GLE1、DDX19A,KRT1、KRT10参与了TNF-α诱导的A549细胞Nox1活化的基因调控,为研究炎症氧化应激细胞模型的Nox1启动子区调控蛋白的生物学功能奠定了实验基础。
Objective To screen the regulatory proteins involved in Noxl promoter activation in a cell model of inflammation and oxidative stress. Methods A cell model of inflammation and oxidative stress was established by stimulating A549 cells with tumor necrosis factor-a （TNF-α）. The differential proteins binding to Noxl promoter were screened by DNA pull-down and the binding proteins were separated by 2D electrophoresis and selected according to the their differential expression levels （with over 1.5-fold changes relative to the control level）. The screened proteins were finally identified by MALDI-TOF/ TOF-MS. Results Seven differentially expressed protein spots （all upregulated in the cell model） were obtained, among which GLE1, DDX19A, KRT1 and KRT10 were identified by mass spectrometry. Conclusion GLE1, DDX19A, KRT1 and KRT10 participate in the activation of Noxl promoter in TNF-α-induced A549 cells, and this result provides new insights into the biological roles of the regulatow proteins of Noxl promoter in inflammation and oxidative stress.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2013年第5期703-707,共5页
Journal of Southern Medical University
基金
国家自然科学基金(81070003)~~
