摘要
目的建立针对以血小板衍生生长因子受体-β(plate-let-derived growth factor receptor-β,PDGFR-β)为靶标的特异及灵敏的细胞模型体系,用于酪氨酸激酶受体(receptor tyro-sine kinase,RTK)抑制剂的筛选和研究。方法构建人的PDGFR-β真核表达载体pcDNA3.1-PDGFR-β,将其转染至NIH 3T3细胞,获得稳定转染的细胞克隆,并对其进行功能学鉴定及应用;应用瞬时转染PDGFR-β的HeLa细胞,建立PDGF依赖性的受体磷酸化细胞模型。结果在无血清培养条件下,稳定转染人PDGFR-β的NIH 3T3细胞获得了PDGF依赖性的细胞增殖特征;HeLa细胞经瞬时转染PDG-FR-β后,可以检测出明显的PDGF依赖性的受体酪氨酸磷酸化。通过应用已知PDGFR-β抑制剂及自行合成化合物,确证了以上细胞模型具有机制针对性特征。结论所构建并确证的细胞模型具有特异性和灵敏性,适用于较高通量的PDGFR-β和其它RTK抑制化合物的体外筛选与研究。
To construct and validate the in vitro cellbased models for the purpose of screening and investigating small molecules targeting PDGFR-13. Methods Plasmid containing the human PDGFR-β gene was constructed and stably transfected into the mouse NIH 3T3 cells,followed by the functional analysis of the transfected clone cells. HeLa cells were transiently transfected with the exogenous PDGFR-β, and the PDGF-dependent tyrosine phosphorylation was examined. Results Clone cells with stable transfection of PDGFR-β retained the PDGF-dependent growth in the absence of serum in culture medium. HeLa cells transiently transfected with PDGFR-13 showed the PDGF-dependent tyrosine phosphorylation. Validation of the mechanism-based cell models above was carried out by the application of known PDGFR-β inhibitors as well as small molecules synthesized in the lab. Coa^lusioa The cell models demon- strated high specificity and sensitivity in terms of PDGF-PDGFR- β pathway, which would serve as mechanism-based cell models for screening small molecules targeting PDGFR-β and other RTKs.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2008年第5期688-691,共4页
Chinese Pharmacological Bulletin
基金
粤港关键领域重点突破资助项目(NoYG2005059)
