高通量筛选JAK-STAT6信号传导通路抑制剂方法的初步建立  

Identification of inhibitors that target JAK-STAT6 signal pathway via high-throughput screening

作　　者：笪宇蓉[1] 姚智[1] 李静雅[2] 邵洁[1] 沈强[2] 东莉洁[1] 李佳[2] 杨洁[1] 

机构地区：[1]天津医科大学免疫教研室,天津300070 [2]中科院上海药物所国家新药筛选中心,上海201203

出　　处：《中国药理学通报》2006年第8期943-947,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No30300070);国家教育部新世纪人才支持计划(NCET-04-0245);高等学校博士学科点专项科研基金资助项目(No20040062003);天津市应用基础研究重点资助项目(No043802811);天津医科大学自然科学基金资助项目(No2005KY01)

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。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-d were measured to optimize the assay condition. 1600 compounds were screened by the method. Results A JAK-STAT6 activation depended cell model was setup and 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.

关 键 词：JAK/STAT6 高通量筛选 工程细胞株 抑制剂 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R392.11[医药卫生—基础医学]