【摘要】 目的构建稳定干扰真核起始因子4E(eIF4E)基因的鼻咽癌CNE2细胞系。方法设计针对eIF4E基因mRNA序列的RNA干扰序列,以慢病毒载体pGC-LV为基础构建感染体系。慢病毒感染CNE2细胞后,经嘌呤霉素压力筛选稳定敲减eIF4E基因的CNE2细胞株。采用定量聚合酶链反应和免疫印迹法检测eIF4EmRNA和蛋白的表达水平;采用细胞增殖和迁徙实验验证eIF4E敲减对CNE2细胞的影响。结果成功构建并筛选获得带有eIF4E特异性短发夹RNA的慢病毒颗粒;将慢病毒颗粒转入CNE2细胞,经抗菌药物压力筛选获得稳定敲减eIF4E基因的CNE2细胞(CNE2siEp)及阴性对照细胞(CNE2siNC)。与CNE2siNC细胞相比,CNE2siEp细胞eIF4EmRNA表达水平下调76.0%,蛋白表达水平下调50.0%,细胞增殖活性下调85.0%,运动活性下调59.0%。结论成功构建稳定敲减eIF4E的CNE2细胞系,其eIF4E基因表达水平与功能明显受抑。更多还原

【Abstract】 Objective To establish a nasopharyngeal carcinoma CNE2 cell line with stable interference of eukaryotic initiation factor 4E(eIF4E)gene.Methods The interference sequences,targeting eIF4 Egene sequence,were designed,coated and transfected into CNE2 cells,by using lentiviral vector infection system based on pGC-LV.The cells containing stable transformants were screened by the ability of resistance to puromycin.The expression of eIF4 E was assayed by real-time polymerase chain reaction and Western Blot.The effects of eIF4 Eknockdown on proliferation and migration of CNE2 cells were tested.Results The lentivirus particles with short hairpin shRNA targeting eIF4 Egene were constructed and screened successfully.CNE2 cells were transfected and anti-pure positive cell,which was named CNE2 siEp cell,were obtained by antibiotic screening.Compared with negative control cells(CNE2siNC),the expression of eIF4 EmRNA and protein in CNE2 siEp cells were significantly decreased by 76.0%and 50.0%respectively.The proliferation and migration of CNE2 siEp cells were also decreased by 85.0% and 59.0% respectively.Conclusion The stable eIF4 Eknockdown cell line(CNE2siEp)was constructed successfully,in which the expression and functions of eIF4 Egene were inhibited significantly.更多还原