EGFR过表达对原代肝细胞AKT的激活作用

EGFR Overexpression Activates the AKT Signaling in Primary Hepatocytes

目的构建含表皮生长因子受体EGFR基因的重组腺病毒载体,并观察所构建腺病毒介导的EGFR过表达对其下游的AKT通路的激活。方法将EGFR的cDNA片段克隆至pshuffle-CMV载体,将该载体与pAdEasy质粒进行细菌内同源重组从而获得重组腺病毒载体pAd-EGFR,之后在Ad293细胞中进行包装以及扩增,并对病毒效价进行检测;最后利用所制备的腺病毒Ad-EGFR感染小鼠原代肝细胞,利用蛋白印迹法Western blot检测EGFR在原代肝细胞中的表达及其对信号分子AKT的活化作用。结果成功制备具有生物学活性的EGFR重组腺病毒,并观察到该重组腺病毒在Huh-7细胞介导EGFR的过表达以及在原代肝细胞中对EGFR下游PI3K-AKT通路的激活作用。结论所构建的EGFR重组腺病毒载体可以介导细胞中EGFR的过表达并激活PI3K-AKT信号通路,为今后研究EGFR的相关生物学功能奠定了基础。

Objective To prepare EGFR recombinant adenovirus, and examine the activation of PI3K - AKT pathway in primary hepatoeytes by EGFR overexpression. Methods Mouse EGFR cDNA was first cloned into the shuttle vector pShuttle - CMV, then re- combined with pAdEasy - 1 plasmid in E. coli strain BJ5183 to obtain the pAd - EGFR vector, which was then transfected into 293A cells for viral package. Huh - 7 cells and mouse primary hepatocytes were infected with adenovirus Ad - EGFR and examined EGFR expression of and AKT phosphorylation by Western blotting analysis. Results We successfully prepared EGFR recombinant adenoviruses, and found that EGFR overexpression activates PI3 K - AKT pathway in primary hepatocytes. Conclusion The established EGFR adenovirus vector may provide a useful tool in investigating the role of EGFR in liver biology.