稳定表达细胞色素P4502B6的Flp-In^(TM)CHO细胞系的建立及鉴定  

Establishment and identification of a Flp-In^(TM)CHO cell line for stable expression of cytochrome P4502B6

作　　者：李靖[1,2] 陆定艳 陈帅帅 孙佳 郑林[1] 李勇军[3] 刘亭[1] LI Jing;LU Dingyan;CHEN Shuaishuai;SUN Jia;ZHENG Lin;LI Yongjun;LIU Ting(State Key Laboratory of Functions and Applications of Medicinal Plants&Guizhou Key Laboratory of Pharmaceutics,Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Pharmacy,People's Hospital of Wanshan District,Tongren 554200,Guizhou,China;Engineering Research Center,Ministry of Education for the Development and Application of Ethnic Medicine,Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学贵州省药物制剂重点实验室&省部共建药用植物功效与利用国家重点实验室,贵州贵阳550004 [2]万山区人民医院药剂科,贵州铜仁554200 [3]贵州医科大学民族药与中药开发应用教育部工程研究中心,贵州贵阳550004

出　　处：《贵州医科大学学报》2024年第10期1435-1439,1446,共6页Journal of Guizhou Medical University

基　　金：国家自然科学基金项目(82360817);贵州省省级科技计划项目(黔科合基础-ZK〔2022〕重点037);贵州省优秀青年科技人才项目(黔科合平台人才〔2021〕5619);贵州省科技计划项目(黔科合中引地〔2023〕006)。

摘　　要：目的构建稳定表达细胞色素P4502B6(CYP2B6)的Flp-In^(TM)CHO细胞系。方法将Flp-In^(TM)CHO细胞分为Flp-In^(TM)CHO组、Flp-In^(TM)CHO空载体组及Flp-In^(TM)CHO-CYP2B6组,通过Lipofectamine2000转染试剂,分别将pcDNA5/FRT空质粒和pcDNA5/FRT-CYP2B6重组质粒转染至Flp-In^(TM)CHO空载体组细胞和Flp-In^(TM)CHO-CYP2B6组细胞中,利用实时荧光定量PCR(qRT-PCR)、蛋白免疫印迹(Western blot)及荧光素酶实验检测转染细胞中CYP2B6 mRNA水平、蛋白表达水平及活性,用四氮唑盐(MTS)测定转染CYP2B6的Flp-In^(TM)CHO细胞对环磷酰胺的毒性敏感性。结果与Flp-In^(TM)CHO组和Flp-In^(TM)CHO空载体组相比,Flp-In^(TM)CHO-CYP2B6组细胞中CYP2B6的mRNA和蛋白表达水平显著升高(P<0.01)、酶活性显著增加(P<0.001),对环磷酰胺的毒性敏感度显著增加(P<0.001)。结论成功构建稳定表达CYP2B6的Flp-In^(TM)CHO细胞系,为研究药物代谢和筛选毒性药物提供工具。Objective To establish the Flp-In^(TM)CHO cell line for stable expression of cytochrome P4502B6(CYP2B6).Methods Flp-In^(TM)CHO cells were divided into three groups:Flp-In^(TM)CHO group,Flp-In^(TM)CHO vector group,and Flp-In^(TM)CHO-CYP2B6 group.By using Lipofectamine 2000 transfection reagent,the pcDNA5/FRT empty vector and the pcDNA5/FRT-CYP2B6 recombinant plasmid were respectively transfected into the cells of the Flp-In^(TM)CHO vector group and the Flp-In^(TM)CHO-CYP2B6 group.The mRNA and protein expression and activity of CYP2B6 were detected by real-time quantitative PCR(qRT-PCR),Western blot and Luciferase experiment.The toxicity sensitivity of Flp-In^(TM)CHO cells transfected with CYP2B6 to cyclophosphamide was determined by MTS.Results Compared with the cells transfected with pcDNA5/FRT empty plasmid,the mRNA and protein expression of CYP2B6 in CYP2B6-CHO were elevated(P<0.01),and the enzyme activity and sensitivity to cyclophosphamide of CYP2B6-CHO also increased(P<0.001).Conclusion The Flp-In^(TM)CHO cell line for stable expression of CYP2B6 has been established successfully,which provides a tool for metabolism research and toxicant screening.

关 键 词：细胞色素P4502B6 稳定表达 酶活性 环磷酰胺 Flp-In^(TM)CHO细胞系 

分 类 号：R966[医药卫生—药理学]