Livin shRNA表达载体的构建及在胰腺癌细胞的效应研究  

Construction of LivinshRNA vector and its effect on expression of Livin mRNA and protein in pancreatic cancer cells

作　　者：张财明[1] 季一鸣[1] 朱敏[1] 吕尚东[1] 王爱东[1] 

机构地区：[1]台州恩泽医疗中心(集团)浙江省台州医院肝胆外科,317000

出　　处：《浙江医学》2015年第9期747-749,761,共4页Zhejiang Medical Journal

基　　金：台州市科技局计划项目(102KY09)

摘　　要：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G4侣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westefn 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。Objective To construct LivinshRNA eukaryotic expression vectors and to investigate its effect on expression 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 Western 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.

关 键 词：LIVIN基因 RNA干扰 转染 PANC-1细胞 

分 类 号：R73-3[医药卫生—肿瘤]