血管内皮生长因子-2抑制剂细胞筛选模型的构建及应用  

Construction and Application of Cell-Based Models for Screening Vascular Endothelial Growth Factor Receptor-2 Inhibitors

作　　者：付水[1,2] 袁远[1] 张苗苗[1] 褚邦勇[1] 宁志强 

机构地区：[1]浙江省台州医院,浙江台州317000 [2]深圳微芯生物科技有限公司,广东深圳518057

出　　处：《中国药学杂志》2011年第24期1869-1873,共5页Chinese Pharmaceutical Journal

基　　金：粤港关键领域重点突破资助项目(YG20905059)

摘　　要：目的建立针对以血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2,VEGFR-2)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 tyrosine kinase,RTK)抑制剂的筛选和研究。方法构建人源VEGFR-2真核表达载体pcDNA3.1-VEGFR-2,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VEGFR-2的HeLa细胞,建立VEGF依赖性的受体磷酸化细胞模型及应用。结果在无血清培养条件下,稳定转染VEGFR-2的NIH 3T3细胞获得VEGF依赖性的细胞增殖特征;HeLa细胞经瞬时转染VEGFR-2后,可以检测出明显的VEGF依赖性的受体酪氨酸磷酸化。通过应用已知VEGFR-2抑制剂及自行合成化合物,确证以上细胞模型具有较高机制针对性的特征。结论所构建并确证的细胞模型具有较高特异性和灵敏性,可适用于VEGFR-2和其他RTK抑制化合物的体外筛选与研究。OBJECTIVE To construct and validate the in vitro cell-based models for the purpose of screening and investigating small molecules targeting VEGFR-2. METHODS Plasmid containing human VEGFR-2 gene was constructed and stably transfected into NIH 3T3 cells, then the function of the transfected clone ceils was analyzed. HeLa cells were transiently transfected with exogenous VEGFR-2, and the VEGF-dependent tyrosine phosphorylation was examined. RESULTS The cloned cells with stable transfection of VEGFR-2 retained VEGF-dependent growth in the absence of serum in culture media. HeLa cells transiently transfected with VEGFR- 2 showed VEGF-dependent tyrosiue phosphorylation. Validation of the above mechanism-based cell models was carried out by applica- tion of known VEGFR-2 inhibitors as well as small molecules synthesized in our lab. CONCLUSION The cell models demonstrated high specificity and sensitivity in terms of VEGF-VEGFR-2 pathway, which would serve as mechanism-based cell models for screening for small molecules targeting VEGFR-2 and other RTKs.

关 键 词：血管内皮生长因子 血管内皮生长因子受体 NIH3T3细胞 HELA细胞 酪氨酸激酶受体 

分 类 号：R73[医药卫生—肿瘤]