细胞色素P4502E1稳定转染Flp-In^(TM)CHO细胞模型的建立  

Construction of Flp-In^(TM)CHO cell model stably transfected with human cytochrome P4502E1

作　　者：赵永龙 郭玮钰 龙昌兰 陆定艳 陈帅帅 李勇军[2] 刘亭 ZHAO Yonglong;GUO Weiyu;LONG Changlan;LU Dingyan;CHEN Shuaishuai;LI Yongjun;LIU Ting(School of Pharmacy,Guizhou Medical University,Guiyang 550004,Guizhou,China;Engineering Research Center for the Development and Application of Ethnic Medicine and TCM(Ministry of Education),Guizhou Medical University,Guiyang 550004,Guizhou,China;Guizhou Provincial Key Laboratory of Pharmaceutics&State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学药学院,贵州贵阳550004 [2]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 [3]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

出　　处：《贵州医科大学学报》2022年第12期1390-1395,共6页Journal of Guizhou Medical University

基　　金：国家自然科学基金(82060703,U1812403-05);贵州省省级科技计划项目资助(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：目的构建稳定表达细胞色素P450家族2亚家族E成员1(CYP2E1)的Flp-In^(TM)CHO细胞系,并进行药物相互作用筛选。方法利用Lipofectamine■2000转染试剂将pcDNA5/FRT-CYP2E1重组质粒和pcDNA5/FRT-空质粒-并转染Flp-In^(TM)CHO细胞,采用潮霉素B(500 mg/L)进行稳定性筛选、聚合酶链式反应(PCR)检测细胞中是否成功插入能够表达CYP2E1酶的目的基因、实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹法(Western blot)检测细胞中CYP2E1 mRNA和蛋白表达,采用对乙酰氨基酚(APAP)检测CYP2E1酶对APAP的毒性敏感性。结果与Flp-In^(TM)CHO空质粒组比较,PCR结果显示Flp-In^(TM)CHO-CYP2E1细胞分别在2000 bp、200 bp左右有CYP2E1酶目的基因的明显条带;qRT-PCR和Western blot结果显示,Flp-In^(TM)CHO-CYP2E1细胞的CYP2E1 mRNA和蛋白表达水平均明显增加(P<0.001);APAP检测结果显示CYP2E1酶对APAP的毒性敏感性也显著增强。结论成功构建了稳定表达CYP2E1的Flp-In^(TM)CHO细胞模型,且表达的CYP2E1酶对APAP的毒性敏感性增强。Objective To Construct Flp-In^(TM)CHO cell line stably expressing cytochrome P450 family 2 subfamily E member 1(CYP2E1)and conduct the drug interaction screening.Methods In this paper,a recombinant plasmid pcDNA5/FRT-CYP2E1 was constructed and the recombinant plasmid pcDNA5/FRT-CYP2E1 and pcDNA5/FRT-empty plasmid were transfected into Flp-In^(TM)CHO cells respectively using Lipofectamine2000 transfection reagent.Hygromycin B(500 mg/L)was used to perform stable screening and detect whether the target gene CYP2E1 has been successfully inserted into the cells by polymerase chain reaction(PCR)and detect the mRNA and protein expression levels of CYP2E1 in the cells by real-time fluorescent quantitative PCR(qRT-PCR)and Western blot.The toxicity sensitivity of CYP2E1 to APAP was determined by acetaminophen(APAP).Results Compared with the empty plasmid group of Flp-In^(TM)CHO,the PCR results showed that Flp In^(TM)CHO-CYP2E1 cells had significant bands of target genes of CYP2E1 enzyme at about 2000 bp and 200 bp respectively;the results of qRT-PCR and Western blot also showed that the mRNA expression level of CYP2E1 and the corresponding protein expression level of CYP2E1 in Flp In^(TM)CHO-CYP2E1 cells were significantly increased(P<0.001),and its toxicity sensitivity to APAP also increased significantly.Conclusion The Flp-In^(TM)CHO cell model stably expressing CYP2E1 can be successfully constructed.

关 键 词：细胞色素P450家族2亚家族E成员1 稳定表达 Flp-In^(TM)CHO细胞 蛋白免疫印迹法 实时荧光定量PCR 转染 

分 类 号：R963[医药卫生—微生物与生化药学]