基于STAT3转录调节的筛药模型的建立

Construction of a drug screening model based on transcription regulation of STAT3

目的 依据造血生长因子信号转导途径中重要转录因子STAT3的转录调节作用建立基于报告基因的靶向STAT3转录调节的筛药模型 ,用此模型筛选具有G CSF样活性的化合物。方法 构建诱导性表达载体 pTKS3 CAT并转染入表达G CSF受体的NFS 6 0不依赖细胞 ,筛选报告基因CAT表达受rhG CSF诱导的阳性克隆 ,采用ELISA法检测化合物对CAT表达活性的影响。同时 ,对模型的特异性和灵敏性进行检测。结果 在转染有重组质粒的NFS 6 0不依赖细胞中 ,CAT表达受rhG CSF诱导并呈剂量依赖关系 ,EC50 等于 0 0 44nmol·L-1,而rhEPO不能诱导CAT表达。结论 以上模型的CAT基因表达活性能够被其特异性配体强诱导表达 ,利用此模型在 96孔板上用ELISA法测定CAT基因的诱导表达水平可筛选具有G CSF样活性的化合物。

AIM To find small molecule organic mimetics of G-CSF, according to the transcription regulating effect of the important transcription factors-STAT3 (signal transducers and activators of transcription3) in hematopoiesis growth factor's signal transductional passageway, we established a drug screening model targeting transcription regulating of STAT3. METHODS A recombinat vector pTKS3-CAT was constructed. Then pTKS3-CAT and pTK-Hyg were transfected into NFS-60 cells expressing G-CSF receptor with lipofectamine 2000. Cells derived from hygromycin-resistant colonies were tested for CAT activity, and positive colonies were isolated. At the same time, the sensitivity and the specialty of the model were tested. RESULTS A dose-dependent expression of CAT gene with half-maximal induction by rhG-CSF at 0.044 nmol·L -1 was observed. After treating with rhEPO CAT activity couldn't be tested. CONCLUSION The expression of CAT gene could be strongly induced by its special ligands in drug screening model. This model can be used to assay CAT from extracts of cells grown with CAT-ELISA method in microtiter wells and then to screen small molecular compounds with G-CSF-like activity.