摘要
目的建立稳定表达人细胞色素P450氧化还原酶(POR)的Flp-In^(TM) CHO细胞系,为进一步建立稳定双表达人POR与人细胞色素P450(CYP)的细胞系奠定基础。方法构建POR重组慢病毒并感染Flp-In^(TM) CHO细胞,荧光显微镜观察绿色荧光蛋白表达,挑选转染细胞进行嘌呤霉素筛选和单克隆培养,以获得稳定转染细胞株。利用丝裂霉素C(MMC)细胞毒实验、实时荧光定量PCR和Western blot法检测细胞POR的表达,获得稳定表达POR的Flp-In^(TM) CHO-POR细胞株。构建稳定双表达POR和CYP2C19的Flp-In^(TM) CHO-POR细胞(Flp-In^(TM) CHO-POR-2C19)和单表达CYP2C19的Flp-In^(TM) CHO细胞(Flp-In^(TM) CHO-2C19),并用环磷酰胺(CPA)测定CYP2C19酶活性。结果与感染阴性对照病毒的Flp-In^(TM) CHO细胞相比,感染POR重组慢病毒的Flp-In^(TM) CHO细胞MMC代谢活性升高,POR mRNA和蛋白的表达水平增加,说明获得了可稳定表达POR的Flp-In^(TM) CHO-POR细胞。与Flp-In^(TM) CHO细胞相比,Flp-In^(TM) CHO-2C19的CPA代谢活性无显著差异,而Flp-In^(TM) CHO-POR-2C19的CPA代谢活性增加且明显高于Flp-In^(TM) CHO-2C19细胞。结论成功建立了稳定表达POR且可进一步用于CYP转基因细胞构建的Flp-In^(TM) CHO-POR细胞系。
Objective To establish a Flp-In^(TM) cCHO celline stably expressing human cytochrome P450 oxidoreductase(POR)to lay a solid foundation for the further construction of cell lines stably co-expressing human POR and human cytochrome P450(CYP).Methods POR recombinant lentivirus was established and infected with Flp-In^(TM) CHO cells,and the expression of green fluorescent protein was observed by fluorescence microscope for monoclonal screening.Mitomycin C(MMC)cytotoxic assay,Western blot analysis and quantitative real-time PCR(qRT-PCR)were employed to detect the activity and expression of POR,and eventually obtained a cell line stably expressing POR(Flp-In^(TM) CHO-POR).Flp-In^(TM) CHO-POR cells(Flp-In^(TM) CHO-POR-2C19)stably co-expressing POR and CYP2C19,and Fip-In^(TM) CHO cells stably expressing CYP2C19(Flp-In^(TM) CHO-2C19)were constructed,and the activity of CYP2C19 was measured by cyclophosphamide(CPA).Results The consequences of MMC cytotoxic assay,Western blot and qRT-PCR illuminated that Flp-In^(TM) CHO cells infected with POR recombinant lentivirus had elevated MMC metabolic activity and boosted the expression of POR mRNA and protein,compared with Flp-In^(TM) CHO clls infected with negative control virus,indicating that Flp-In^(TM) CHO-POR cells stably expressing POR were obtained.No significant disparity existed in the metabolic activity of CPA between Flp-In^(TM) CHO-2C19 and Flp-In^(TM) CHO cells,whereas the metabolic activity enhanced in Flp-In^(TM) CHO-POR-2C19 and was significantly higher than in Flp-In^(TM) CHO-2C19 cells.Conclusion The stable expression of Flp-In^(TM) CHO-POR cell line is successfully established and can be further applied to construct CYP transgenic cells.
作者
刘欢
刘亭
陆定艳
孙莉
何俊奇
李勇军
王永林
孙佳
席晓岚
LIU Huan;LIU Ting;LU Dingyan;SUN Li;HE Junqi;LI Yongjun;WANG Yonglin;SUN Jia;XI Xiaolan(Guizhou Provincial Key Laboratory of Pharmaceutics,State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550004;Department of Pharmaceutical Analysis,School of Pharmacy,Guizhou Medical University,Guiyang 550025;Engineering Research Center for the Development and Application of Ethnic Medicine and TCM(Ministry of Education),Guizhou Medical University,Guiyang 550004,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2023年第2期103-108,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(82060703,U1812403-5)
贵州省省级科技计划项目(黔科合基础-2K[2022]重点037)
贵州省普通高等学校科技拔尖人才项目(黔教合KY字[2021]033)
贵州省优秀青年科技人才项目(黔科合平台人才[2021]5619号)。
