人EGFR显性负性突变体负调控内源性EGFR功能的机制分析  

Analysis of the Mechanism Underlying the Inhibitory Effect of Human Dominant Negative Epidermal Growth Factor Receptor on Endogenous Epidermal Growth Factor Receptor Function

作　　者：廖刚[1] 王子卫[1] 赵林[1] 张能[1] 董浦江[2] 

机构地区：[1]重庆医科大学附属第一医院胃肠外科,中国重庆400016 [2]重庆医科大学附属第一医院实验研究中心,中国重庆400016

出　　处：《生命科学研究》2010年第3期203-207,239,共6页Life Science Research

基　　金：国家自然科学基金资助项目(30972872)

摘　　要：通过定向克隆法构建真核表达载体pEGFPN1-DNEGFR,脂质体介导下转染体外培养的SGC-7901细胞,应用Western blotting检测DNEGFR-EGFP蛋白的表达,激光共聚焦显微镜对DNEGFR-EGFP亚细胞结构定位检测;并经RT-PCR、Western blotting检测DNEGFR-EGFP对内源性EGFRmRNA水平、蛋白及磷酸化水平的影响.成功检测到DNEGFR-EGFP蛋白的表达,DNEGFR-EGFP蛋白主要定位于细胞膜,DNEGFR-EGFP能降低内源性EGFR蛋白磷酸化水平,而对内源性EGFRmRNA水平及蛋白水平无影响.研究证明DNEGFR通过降低内源性EGFR蛋白磷酸化水平负调控EGFR功能,为靶向EGFR显性负性策略在肿瘤生物治疗中的进一步研究打下基础.To construct the eukaryotic expression vector（pEGFPN1-DNEGFR）of human dominant negative epidermal growth factor receptor（DNEGFR）,then to explore the mechanism of the inhibitory effect of human DNEGFR on endogenous epidermal growth factor receptor（EGFR） function.The pEGFPN1DNEGFR was constructed by directional cloning,then transfected into SGC-7901 cells,mediated by Lipofectamine 2000.The expression and the subcellular localization of DNEGFR-EGFP in SGC-7901 cells were detected by Western blotting and laser confocal microscope respectively.The influence of DNEGFREGFP on endogenous mRNA,protein and protein phosphorylation levels of EGFR was detected by reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting respectively.The pEGFPN1DNEGFR was constructed successfully.The expression of DNEGFR-EGFP,and its predominant localization on the cell membrane of SGC-7901 cells were identified.DNEGFR-EGFP decreased the phosphorylation level of EGFR protein,but didn＇t change EGFR mRNA or protein level.The successful construction of pEGFPN1-DNEGFR and the confirmation of the inhibitory effect of DNEGFR on endogenous EGFR protein phosphorylation level,laid a solid foundation for further research on EGFR-targeted dominant negative strategy in cancer biotherapy.

关 键 词：表皮生长因子受体(EGFR) 显性负性突变体 磷酸化水平 定向克隆 亚细胞结构定位 

分 类 号：Q78[生物学—分子生物学]