稳定表达CYP2A13和CYP2A13/POR的Flp-In CHO重组细胞系的构建  

Establishment of Flp-In CHO recombinant cell lines stably expressing CYP2A13 and CYP2A13/POR

作　　者：孙莉 陆定艳 刘欢[1,2] 何俊奇 孙佳 王永林[1] 李勇军[3] 杨畅[1] 刘亭[1] SUN Li;LU Ding-yan;LIU Huan;HE Jun-qi;SUN Jia;WANG Yong-lin;LI Yong-jun;YANG Chang;LIU Ting(Guizhou Provincial Key Laboratory of Pharmaceutics/State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550004,China;School of Pharmacy,Guizhou Medical University,Guiyang 550025,China;Engineering Research Center for the Development and Application of Ethnic Medicine and TCM(Ministry of Education),Guizhou Medical University,Guiyang 550004,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,贵阳550004 [2]贵州医科大学药学院,贵阳550025 [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,贵阳550004

出　　处：《中国药理学通报》2022年第5期795-799,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81603189,U1812403-05);贵州省优秀青年科技人才项目(黔科合平台人才[2021]5619号);贵州省普通高等学校科技拔尖人才项目(黔教合KY字[2021]033)。

摘　　要：目的分别构建稳定表达细胞色素P450家族2亚家族A成员13(cytochrome P450 family 2 subfamily A member 13,CYP2A13)的Flp-In CHO细胞系(CYP2A13-CHO)和稳定表达CYP2A13和细胞色素P450氧化还原酶(POR)的Flp-In CHO细胞系(CYP2A13-POR-CHO),并从中筛选代谢活性较好的细胞系。方法课题组前期通过慢病毒转染构建了稳定表达POR的Flp-In CHO细胞系(POR-Flp-In CHO)。该文构建了pcDNA5/FRT-CYP2A13重组质粒,利用Lipofectamine^(TM) 2000转染试剂将pcDNA5/FRT-CYP2A13重组质粒分别转染到Flp-In CHO细胞和POR-Flp-In CHO细胞中。通过荧光定量PCR(qRT-PCR)、Western blot和黄曲霉素B1(AFB1)/4-甲基亚硝胺-1-3-吡啶基-1-丁酮(NNK)细胞毒实验来检测CYP2A13的表达及其活性,并比较了CYP2A13-CHO和CYP2A13-POR-CHO两种重组细胞系的代谢活性。结果与未转染的细胞相比,CYP2A13-CHO和CYP2A13-POR-CHO的CYP2A13 mRNA和蛋白的表达量均明显增加。且与CYP2A13-POR-CHO相比,CYP2A13-CHO细胞对AFB1、NNK的敏感度更高。结论建立了稳定表达CYP2A13且代谢活性较好的CYP2A13-CHO细胞系,为后续筛选能被CYP2A13代谢活化的前致癌物提供了工具。Aim To construct Flp-In CHO cell line(CYP2A13-CHO)stably expressing cytochrome P450 family 2 subfamily A member 13(CYP2A13)and Flp-In CHO cell line(CYP2A13-POR-CHO)stably co-expressing CYP2A13 and cytochrome P450 oxidoreductase(POR),from which a cell line with better metabolic activity is selected.Method In our previous study,we had constructed a Flp-In CHO cell line(POR-Flp-In CHO)stably expressing POR using lentiviral vector.The recombinant plasmids of pcDNA5/FRT-CYP2A13 were constructed and transfected into Flp-In CHO cells and POR-Flp-In CHO cells through Lipofectamine^(TM) 2000.The expression and activity of CYP2A13 were detected by real-time quantitative PCR(qRT-PCR),Western blot and Aflatoxin B1(AFB1)/4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)cytotoxicity assay and the metabolic activity was compared between CYP2A13-CHO and CYP2A13-POR-CHO.Results Compared with non-transfected cells,the mRNA and protein expression of CYP2A13 in CYP2A13-CHO and CYP2A13-POR-CHO cells both increased significantly.Besides,compared with CYP2A13-POR-CHO,CYP2A13-CHO cells were more sensitive to AFB1 and NNK.Conclusions The Flp-In CHO cell line stably expressing CYP2A13 and with better metabolic activity has been established successfully,which provides a tool for screening of pre-carcinogens that can be metabolically activated by CYP2A13.

关 键 词：细胞色素P450家族2亚家族A成员13 细胞色素P450氧化还原酶 稳定表达 Flp-In CHO细胞 代谢活性 前致癌物 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R345.99[医药卫生—基础医学]