【摘要】 目的构建可用于高通量筛选JAK/STAT6信号传导通路抑制剂的工程细胞株,建立稳定可靠的筛选方法。方法利用基因重组和转染技术,将STAT6特异性识别启动子IgE基因序列和虫荧光素酶报告基因联合插入pCMV质粒,脂质体法转染至HeLa细胞,经潮红霉素B抗性筛选及报告基因检测,得到稳定表达虫荧光素酶的工程细胞株。通过优化溶剂DMSO浓度,IL4作用浓度及孵育时间等筛选条件,建立了可靠的筛选方法,并在此基础上对1600种化合物进行了筛选。结果建立的筛选方法稳定可靠,系统Z′因子达到0.64。通过对1600种化合物的筛选,得到3个抑制效果较理想的化合物并测得其IC50值。结论所建立的高通量筛选方法可用于JAK/STAT6信号传导通路抑制剂的筛选。更多还原

【Abstract】 Aim To establish the stable cell line which expresses luciferase depending on STAT6-mediated gene transcriptional activation. The cell line is used as a model to screen compounds that inhibit JAK- STAT6 signal transduction pathway. Methods The sequence of IgE promoter and luciferase reporter gene were subcloned into the vector to generate recombinant plasmid, which can express luciferase after IL-4 stimulation. The recombinant plasmid was cotransfected into HeLa cells together with hygromycin B DNA. The positive cell clones were selected in the culture media which contains hygromycin B. Some factors such as final concentration of DMSO and working concentration of IL-4 were measured to optimize the assay condition. 1600 compounds were screened by the method. Results A JAK-STAT6 activation depended cell model was setupand a reliable method was established to perform high throughput screening. Z′-factor value of the system was near 0.64. Three compounds were screened to have suppressive effect on JAK-STAT6 pathway. Conclusion This assay can be applied to identify inhibitors targeting JAK-STAT6 signal transduction pathway.更多还原