不同细胞中STAT元件对刺激因子的特异性应答

Stimulating Factors Induced Specific STAT Element Binding Response in Different Cell Lines

以高通量药物筛选为目的,构建转录因子STAT元件驱动的萤火虫荧光素酶报告载体,并进行功能初步验证.人工合成含IRF-1和C-FOS基因上游调控序列中的2个STAT元件的DNA片段,克隆于pGL3-promoter报道质粒作为报道载体pSTAT-luc;为检测其对不同有效因子刺激的反应性,该报道载体与荧光内参照载体pRL-SV40瞬时共转染不同细胞,在各种因子刺激条件下,双荧光素酶检测系统测定化学发光强度.结果显示,pSTAT-luc的报道基因载体符合预期设计;该载体转染细胞实验显示,在STAT途径阳性的HeLa、A549和MCF-7细胞中,特异刺激因子INF-γ和抑瘤素OSM处理能够剂量依赖性的引起细胞内萤火虫荧光素酶的升高;在STAT途径阴性的PC-12细胞,上述因子不能引起荧光素酶的活性改变;以非该途径因子刺激时,HeLa、A549和MCF-7细胞未见发光水平的改变.结果表明,构建的报道系统具有阳性细胞反应特异性以及阳性刺激-反应特异性,能够用于建立靶向STAT信号通路的高通量药物筛选平台.

In purpose of establishing a high-throughput drug screening method,a luciferase reporter vector driven by human STAT（signal transduction and activators of transcription） binding elements were constructed,and the responsiveness upon stimulation were validated in different cell lines.Two STAT binding elements from upstream regulatory sequences of IRF-1 and C-FOS gene were synthesized and cloned into pGL3-Luc-promoter vector.The constructed vector pSTAT-luc,and a inner calibration vector pRL-SV40,were transiently co-transfected into different cell lines and stimulated by various factors.The luciferase activity was assayed using dual-luciferase system to validate cell specific response.The result showed that in STAT pathway positive HeLa,A549 and MCF-7 cell lines,STAT specific factors INF-γ and OSM strongly activated pSTAT-luc reporter in dose-dependent way.Neither stimulation with irrelevant factors in STAT pathway positive cells,nor with STAT specific factors in STAT pathway negative cell line PC-12,produced positive response.The result demonstrated that this vector was highly cell and pathway specific for STAT pathway activation and can be used tools for high-throughput drug screening based on STAT signal pathway.