含表皮生长因子受体(EGFR)启动子萤光素酶报告基因的三阴性乳腺癌细胞模型的建立

Constraction of triple negative breast cancer cell model containing EGFR promoter and luciferase reporter gene

目的建立稳定表达人表皮生长因子受体(EGFR)启动子及萤光素酶(Luc)报告基因的三阴性乳腺癌细胞系,并对其应用进行初步验证。方法采用基因重组技术,构建含有EGFR启动子以及Luc报告基因的慢病毒载体,并感染MDA-MB231细胞,经过嘌呤霉素筛选获得能稳定表达Luc活性的MDA-MB231-EGFR-Luc2细胞系;分别采用EGFR激活剂EGF以及抑制剂吉非替尼处理细胞后检测Luc活性变化。结果通过基因测序和质粒双酶切鉴定,EGFR基因启动子Luc报告基因-慢病毒表达载体构建成功,经过嘌呤霉素筛选获得稳定表达Luc的MDA-MB231-EGFR-Luc2细胞;EGF可剂量依赖性的增加细胞中Luc的活性,而吉非替尼则相反。结论建立了MDA-MB231-EGFR-Luc2稳定表达EGFR启动子及Luc报告基因细胞系,为高通量筛选靶向EGFR的抗肿瘤药物提供一类新的细胞模型。

Objective To establish a triple negative breast cancer cell line stably expressing human epidermal growth factor receptor(EGFR)promoter and luciferase(Luc)reporter gene and to preliminarily verify its application.MethodsUsing genetic recombination technology,the lentiviral vector carrying Luc reporter and EGFR promoter sequence was designed and constructed to infect MDA-MB231 cells and obtain MDA-MB231-EGFR-Luc2 cell lines by the selection with puromycin.The Luc luminescence value after stimulating with EGFR activator EGF or inhibitor gefitinib regulating the EGFR promoter activities was detected.Results Gene sequencing and enzyme digestion verified that the lentiviral expression vector carrying Luc reporter vector recombined with EGFR promoter was successfully constructed.Lentivirus-infected MDA-MB231 cells were screened by puromycin,the MDA-MB231-EGFR-Luc2 cells stably expressing firefly Luc was obtained.EGF increased the Luc luminescence value of MDA-MB231-EGFR-Luc2 cells in a dose-dependent manner,while gefitinib did the opposite.Conclusion The cell line of MDA-MB231-EGFR-Luc2 containing EGFR promoter and Luc reporter gene has been successfully constructed,which provides a new cell model for high throughput screening of EGFR-targeting drugs.