细胞色素P4503A46基因的异源表达及药物代谢活性分析

发布时间：2018-08-06 15:35

【摘要】：为了克隆巴马小型猪细胞色素P4503A46基因,建立P4503A46稳定表达肝癌细胞株(Hep G2-P4503A46),并探究其药物代谢活性,通过RT-PCR从肝组织中钓取基因P4503A46,将基因与真核表达载体pc DNA3.1连接并转染Hep G2细胞,经G418筛选10代,获得稳定表达重组细胞株Hep G2-P4503A46,以P4503A特异性代谢底物硝苯地平(NF)探针药物,将探针药物与重组细胞在优化的细胞浓度、药物浓度、孵育时间等条件下进行体外孵育,通过高效液相色谱仪检测其药物代谢(抑制)动力学参数,并将对照组进行比较分析。结果表明,本研究成功从人Bama小型猪肝组织克隆了P4503A46基因,并通过G418筛选,成功建立了细胞稳定表达株,其硝苯地平代谢活性显著高于阴性对照组(P0.01);因此,成功克隆、并建立了小型猪P4503A46的稳定表达细胞株(Hep G2-P4503A46),该细胞株具有显著的硝苯地平代谢活性。
[Abstract]:In order to clone the cytochrome P4503A46 gene of Bama miniature pig, to establish P4503A46 expressing hepatoma cell line (Hep G2-P4503A46) stably, and to explore its drug metabolism activity, the gene P4503A46 was isolated from liver tissue by RT-PCR, and the gene was ligated with eukaryotic expression vector PC DNA3.1 and transfected into Hep G2 cell. The recombinant cell line Hep G2-P4503A46 was screened by G418 for 10 passages. The recombinant cell line Hep G2-P4503A46 was used as a specific metabolic substrate of P4503A, nifedipine (NF) probe drug was used to optimize the cell concentration and drug concentration. The pharmacokinetic parameters were measured by high performance liquid chromatograph (HPLC) and compared with the control group. The results showed that the P4503A46 gene was cloned successfully from human Bama small pig liver tissue, and the stable expression strain was successfully established by G418 screening. The metabolic activity of nifedipine was significantly higher than that of negative control group (P0.01). The stable expression cell line (Hep G2-P4503A46) of miniature pig P4503A46 was established. The cell line showed significant metabolic activity of nifedipine.
【作者单位】：泸州职业技术学院生物医学工程研究所;第三军医大学实验动物中心;
【基金】：国家“十五”科技攻关计划重点项目资助课题(2004BA717B23)
【分类号】：R965

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