Livin shRNA 表达载体的构建及在胰腺癌细胞的效应研究

摘要

Objective To construct LivinshRNA eukaryotic expression vectors and to investigate its effect on ex-pression of Livin mRNA and protein in pancreatic cancer cel line panc- 1. Methods Livin gene interference sequence was synthesized and inserted into pGCsilencerTM U6/Neo/GFP/RNAi vector,which was confirmed by sequencing. The recombi-nant RNAi vector was transfected into pancreatic cancer panc- 1cel s by Lipofectamine 2000. The cel s containing stable transformants were selected by G418, and isolated with limited dilution. The mRNA expression of Livin in the selected clones was detected by RT- PCR, the protein expression of Livin was detected by Western blot. Results The successful construction of RNAi eukaryotic expression vectors targeting Livin was confirmed by sequencing, which was expressed in panc- 1 cel s after transfection as showed by green fluorescence under inverted fluorescence microscope. RT- PCR and West-ern blot revealed that the expression of Livin mRNA and protein was down- regulated in panc- 1 cel s. Conclusion Livin shRNA eukaryotie expression vector was successful y constructed and the transfected pancreatic cancer panc- 1 cel s show a down- regulated expression of Livin mRNA and protein.%目的：构建Livin shRNA真核表达载体，探讨干扰质粒稳定转染的胰腺癌panc-1细胞系效应。方法合成Livin基因干扰序列并定向插入到RNA干扰（RNAi）真核表达载体pGCsilencerTM U6/Neo/GFP/RNAi质粒，通过测序进行鉴定。干扰质粒经脂质体Lipofectamine 2000介导转染panc-1细胞。经G418持续压力选择和有限稀释法获得稳定转染的细胞系。定量RT- PCR检测所筛选克隆Livin mRNA的转录水平，采用western blot验证Livin蛋白表达水平改变。结果测序证明Livin干扰序列及读码框正确，干扰质粒稳定转染的panc-1细胞在倒置荧光显微镜下呈明亮绿色荧光，定量RT- PCR和western blot检测结果显示Livin shRNA序列对胰腺癌细胞系Livin mRNA及蛋白表达均有抑制作用。结论成功构建Livin shRNA真核表达载体，建立Livin shRNA质粒稳定转染的panc-1细胞系可为进一步研究Livin在胰腺癌细胞中的作用奠定基础。