高表达FGFR4细胞株的建立及其生物活性分析  

Establishment of FGFR4 over-expression cell line and analysis of its biological activity

作　　者：黄静[1] 安红丽[2] 张涛[1] 

机构地区：[1]西安交通大学医学院,药物研究所,陕西西安710061 [2]西安交通大学医学院,第一附属医院转化医学中心,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2014年第2期271-275,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.81202495,81101725);中央高校基本科研业务费专项资金(No.xjj2011092)~~

摘　　要：目的构建FGFR4高表达细胞,研究FGFR4对细胞增殖能力的影响及相关蛋白活化机制,为研究以FGFR4为靶点的药物提供细胞模型。方法构建FGFR4真核表达载体并转染HEK293细胞,筛选稳定表达FGFR4细胞株,免疫印迹和免疫荧光分析FGFR4表达水平,细胞计数检测FGFR4高表达细胞的增殖情况,流式细胞仪检测FGFR4对细胞周期的影响,同时对ERK1/2磷酸化水平进行免疫印迹分析。结果在相同培养环境下,HEK293-FGFR4细胞同HEK293-pcDNA细胞相比,其增殖能力提高。周期分析表明,FGFR4使G0/G1期降低,促进细胞的增殖。同时,对MAPK信号通路进行了初步分析,发现FGFR4蛋白可以引起ERK1/2的磷酸化,从而导致MAPK/ERK1/2通路的激活,说明FGFR4可激活MAPK信号通路。结论成功构建了FGFR4高表达细胞株,为筛选以FGFR4为靶标的抗肿瘤药物奠定了前期实验基础。Objective To construct the fibroblast growth factor receptor 4 （FGFR4） over-expression cell line and investigate the effects of FGFR4 on cell proliferation and mechanism of FGFR4-related protein activation so as to provide a molecular model for studying anti-tumor drug candidate targeting on FGFR4. Methods HEK293 cells were transfected with FGFR4-pcDNA plasmid. Then G418-resistant clones of HEK293 cells were obtained under G418 selection. The expression level of FGFR4 was confirmed by immunoblot and immunofluorescence. Cell proliferation rate was measured using cell counting assay. Flow cytometry analysis was performed to explore the effects of FGFR4 on HEK293 cell cycle. ERK1/2 and p-ERK1/2 expression levels were confirmed by immunoblot. Results FGFR4 promoted the proliferation of HEK293 cells as compared with native cells under the same culture condition. Cell phase of G0/G1 was depressed by FGFR4 activation. The FGFR4 proteins elicited constitutive phosphorylation of ERK1/2, leading to the activation of the mitogen-activated MAPK. Conclusion We have successfully constructed the over-expression FGFR4 cell line, which lays an experimental foundation for screening anti-tumor drug candidate targeting on FGFR4.

关 键 词：成纤维细胞生长因子受体4(FGFR4) HEK293 高表达 细胞增殖 

分 类 号：Q71[生物学—分子生物学]