基于JAK/STAT信号通路的高通量药物筛选细胞模型的建立

Establishment of high-throughput drug screening cell models based on JAK-STAT signal pathway

目的 建立基于JAK STAT信号通路的高通量药物筛选细胞模型 ,筛选调节免疫系统、造血系统和抗肿瘤药物。方法 构建以串连的多个STAT的DNA结合序列为调控序列 ,以荧光酶基因作为报告基因的表达载体。通过将该载体分别转入体外培养的不同细胞系中使之稳定表达 ,建立具有激活STAT活性的药物筛选模型 ,并利用多个抗过敏和抗肿瘤药物样品进行检测。结果 建立多株药物筛选细胞模型 ,筛选方法简便 ,操作容易 ,灵敏度高 ,结果稳定。

Aim To discover new drugs which may be applied to diseases of the immune system, hemogenesis system diseases and tumors, several high-throughput drug screening cell models based on JAK-STAT signal pathway have been established. Methods Four repeats of STAT DNA binding conserved sequences were synthesized, subcloned into pGL-Luc reporter vector and stably transfected into cell lines in vitro . Cell clones with high copy numbers of STAT binding sites and reporter genes were chosen as high-throughput drug screening cell models. The cell models were tested with known anti-allergic drugs and anti-tumor drugs by determining luciferase activity. The reaction was performed in 96 well micro-plates with a final volume of 50 μL. Results The cell models by performing rapid fluorescence assay were shown to be highly sensitive and stable after testing with cytokine and drugs. The modification of the expression plasmid simplified this method and made it more practical. It also provided good linear correlation, wide range of assay, highly sensitive and good reproducibility. Conclusion The method can be performed by high-throughput drug screening for effective extraction of Chinese traditional herbs.