利用人工miRNA技术建立甲胎蛋白稳定沉默肝癌细胞及沉默效果评价  

Establishment of Alpha Fetoprotein Stable Knock-down Hepatocarcinoma Cell Line by Artificial miRNA and Evaluation of the Silencing Effect

作　　者：谢佩雯[1,2] 张秀娟[2] 黄海[2] 王学军[2] 王升启[2] 

机构地区：[1]中南大学药学院,长沙410000 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《医学综述》2013年第10期1865-1868,1921,共4页Medical Recapitulate

基　　金：国家科技重大新药创制专项(2012ZX09103301-044)

摘　　要：目的通过建立靶向甲胎蛋白(AFP)基因的人工microRNA(amiRNA)稳定表达HepG2细胞来评价AFP-amiRNA的沉默效果。方法构建靶向AFP基因的amiRNA慢病毒表达质粒,将其转入HEK293T细胞包装为慢病毒并感染HepG2细胞,嘌呤霉素筛选2周后采用半定量反转录聚合酶链反应(RT-PCR)、间接免疫荧光及AFP定量试剂盒检测amiRNA对AFP的抑制效果。结果成功构建靶向AFP的amiRNA慢病毒表达质粒,并建立稳定表达AFP-amiRNA的HepG2细胞,RT-PCR、间接免疫荧光与上清AFP定量结果显示稳转细胞AFP表达水平显著降低。结论高效稳定表达AFP-amiRNA的HepG2细胞株建立成功,并能有效抑制AFP的表达。Objective To establish hepatocarcinoma（HepG2） cell lines with stable expression of artificial miRNA（amiRNAs） targeted alpha fetoprotein（AFP） gene and to evaluate the silencing effect of amiRNAs.Methods Lentiviral amiRNAs expression plasmids targeted at AFP were constructed.Lentivirus were packaged in HEK293T cells to infect HepG2 cells.The stably transducted cell lines were screened by puromycin.The mRNA level of AFP was examined by reverse transcription-polymerase chain reaction（RT-PCR）,and the expression of AFP protein was assayed by indirect immunofluorescense assay and ELISA.Results DNA sequencing showed that the lentiviral amiRNAs expression plasmids targeted AFP gene were successfully constructed.AFP-amiRNAs stable expression cell lines of HepG2 were successfully established by lentivirus infection.The results of RT-PCR,indirect immunofluorescense assay and ELISA demonstrated that the expression of AFP was remarkably reduced both in mRNA and protein levels in AFP-amiRNA stably transducted HepG2 cell lines.Conclusion AFP-amiRNA HepG2 stable cell lines are established and the expression of AFP is reduced both on mRNA and protein levels.

关 键 词：人工miRNA 甲胎蛋白 慢病毒 HEPG2细胞 

分 类 号：Q786[生物学—分子生物学] R735.7[医药卫生—肿瘤]