期刊文献+

ABCC2基因过表达细胞株的建立及鉴定 被引量:1

Construction and identification of the recombinant cell line over-expressing ABCC2 gene
下载PDF
导出
摘要 目的构建携带ABCC2基因的慢病毒载体,转染HEK293细胞,筛选出稳定过表达ABCC2基因的细胞株并进行鉴定,为体外实验确定与多药耐药相关蛋白2(MRP2)外排转运相关的底物药物及其转运机制提供细胞模型。方法根据Gen Bank提供的ABCC2基因c DNA序列设计引物,PCR扩增该基因并将其连接至慢病毒载体PZE-LV105,包装病毒并感染HEK293细胞。用嘌呤霉素进行筛选得到过表达ABCC2基因的稳转细胞株,通过基因测序、实时荧光定量PCR(RTQ-PCR)和蛋白免疫印迹(Western blot)对稳转细胞进行鉴定。结果测序结果证明重组慢病毒过表达载体所携带的ABCC2基因序列与GenBank提供的ABCC2序列一致;RTQ-PCR分析结果显示转染了ABCC2基因的HEK293细胞中MRP2的mRNA相对表达比正常HEK293细胞和空白载体对照HEK293细胞高出约320倍;蛋白免疫印迹试验显示,转染了ABCC2基因的HEK293细胞中MRP2蛋白表达水平比正常HEK293细胞和空白载体对照HEK293细胞高出约150倍。结论该实验成功构建了稳定过表达ABCC2基因的细胞株,该细胞株可用于初步筛选确定MRP2的特异性外排转运底物及其转运机制。 Objective To construct the lentiviral vecter carrying human ABCC2 gene,then to screen out the stable cell line over -ex-pressing ABCC2 after package and transfection to HEK293 cells,thus to provide cell model to determine substrate drugs of multidrug re-sistance protein 2(MRP2)in vitro.Methods The primers were designed according to the cDNA sequence of ABCC2 gene from Gen-Bank.The gene were amplified by PCR and connected to lentiviral vector PEZ -LV105.The recombined lentiviral vector was pack-aged and transfected to HEK293 cells.After puromycin screening,HEK293 cells over -expressing human ABCC2 gene were selected and cloned.Finally,gene sequencing,real -time quantitative PCR(RTQ -PCR)and Western blot assays were performed for identifica-tion.Results RTQ -PCR showed that the ABCC2 mRNA in HEK293 cells transfected with exogenous ABCC2 gene was about 320 times higher than that of normal HEK293 cells and blank carrier HEK293 cells.MRP2 protein level in HEK293 cells with ABCC2 gene transfection was nearly 150 times higher than that of normal HEK293 cells and blank carrier HEK293 cells according to the Western blot.Conclusions Stable recombinant cell line over -expressing ABCC2 was successfully generated.The cell line could be useful in the confirmation of substrate drugs of multidrug resistance protein 2(MRP2)and the transport mechanism in vitro.
出处 《安徽医药》 CAS 2016年第3期433-436,共4页 Anhui Medical and Pharmaceutical Journal
关键词 ABCC2基因 过表达 慢病毒载体 HEK293细胞 ABCC2 gene over -expression lentiviral vector HEK293 cell
  • 相关文献

参考文献14

  • 1Bandler PE, Westlake C J, Grant CE, et al. Identification of regions required for apical membrane localization of human muhidrug re- sistance protein 2 [ J ]. Mol Pharmaco1,2008 ,74 :9 - 19. 被引量:1
  • 2Matsushima S, Maeda K, Hayashi H, et al. Involvement of multiple efflux transporters in hepatic disposition of fexofenadine [ J ]. Mo] Pharmaco1,2008,73 : 1474 - 1483. 被引量:1
  • 3Vasilyeva A,Durrnus S,Li L,et al. Hepatocellular shuttling and recir- eulation of sorafenib - glucuronide is dependent on Abcc2,Abcc3 ,mad Oatpla/lb[J]. Cancer Research,2015,75(13) :2729 -2736. 被引量:1
  • 4Franke RM, Lancaster CS, Peer C J, et al. Dependence of erythro- mycin metabolism on ABCC2 (MRP2) transport function[ J]. Clin Pharmaeol Ther,2011,89 ( 5 ) :693 - 701. 被引量:1
  • 5Vlaming ML, Mohrmann K, Wagenaar E, et al. Carcinogen and an- tieancer drug transport by Mrp2 in vivo: studies using Mrp2 ( Abcc2 ) knockout mice[J]. J Pharmacol Exp Ther, 2006,318 : 319 - 327. 被引量:1
  • 6Kranz J, Hessel S,Aretz J, et al. The role of the efflux carriers Ab- eg2 and Abec2 for the hepatobiliary elimination of benzo [ ct ] py- rene and its metabolites in mice[ J]. Chemico - Biological Interac- tions, 2014,224 : 36 - 41. 被引量:1
  • 7Keppler D. Muhidrug resistance proteins (MRPs, ABCCs) :impor- tance for pathophysiology and drug therapy [ J ]. Handb Exp Phar- macol,2011 (201) :299 - 323. 被引量:1
  • 8Klaassen CD, Aleksunes LM. Xenobiotic, bile acid, and cholesterol transporters: function and regulatiort [ J ]. Pharmaco~ Rev, 2010, 62:91 -96. 被引量:1
  • 9Perez TR. Multidrug resistance :retrospect and prospects in anticancer drug treatment [ J ]. Curt Med Chem,2006,13 : 1859 - 1876. 被引量:1
  • 10Yin W,Xiang P, Li Q. Investigations of the effect of DNA size in transient transfection assay using dual luciferase system [J]. Ana- lytical Biochemistry,2005,346 (2) :289 - 294. 被引量:1

同被引文献14

引证文献1

二级引证文献2

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部