转染重组质粒pBudCE4.1-CYP3A4-GSTA1的肝细胞系的建立及功能评价  

Construction and functional evaluation of a liver cell line tranfected with a vector expressing cytochrome P450 3A4 and glutathione-S-transferase A1

作　　者：常彬霞[1] 貌盼勇[1] 游绍莉[1] 李保森[1] 辛绍杰[1] 

机构地区：[1]中国人民解放军第302医院,北京市100039

出　　处：《世界华人消化杂志》2012年第13期1081-1087,共7页World Chinese Journal of Digestology

基　　金：中国人民解放军军队临床高新技术重大基金资助项目;No.2010gxj097

摘　　要：目的:获得一株适合用于生物人工肝支持系统的细胞材料.方法:用已构建好的重组质粒pBudCE4.1-CYP3A4-GSTA1转染肝脏肿瘤细胞系C3A,用Zeocin筛选,所得细胞系命名为C3A-未优势化;通过qRT-PCR方法检测目的基因表达情况;对转染重组质粒的肝脏肿瘤细胞系C3A的合成、代谢、解毒等功能及性状进行综合评价.结果:成功构建了转染重组质粒pBudCE4.1-CYP3A4-GSTA1的细胞系;构建好的C3A-未优势化细胞系通过qRT-PCR方法检测其目的基因CYP3A4和GSTA1的表达量较正常C3A细胞系高;通过色谱法证明CYP3A4活性较正常C3A细胞系高;用免疫组织化学实验证实目的基因GSTA1的表达较正常C3A细胞系多;另外,对利多卡因的代谢能力亦高于正常C3A细胞.结论:构建的C3A-未优势化细胞系功能有所改善,有望成为生物人工肝系统的细胞材料.AIM： To generate a new cell strain that could be used in the bioartificial liver support system. METHODS： The C3A cell line was transfected with the recombinant plasmid pBudCE4.1-CYP 3A4-GST A1, which expresses both cytochrome P450 3A4 （CYP 3A4） and glutathione-S-transferase A1 （GSTA1）, and cultured in MEM containing 400 mg/L Zeocin for 2 wk. The obtained cell line was named C3A-Unoptimized. The expression of CYP 3A4 and GSTA1 in C3A-Unoptimized cells was detected by qRT-PCR, and the function of the C3A-Unoptimized cell line was evaluated. RESULTS： The C3A-Unoptimized cell line stably expressed both CYP 3A4 and GST A1.The expression levels of CYP 3A4 and GSTA1 were higher in C3A-Unoptimized cells than in non-transfected C3A cells. Chromatogram assay showed that the activity of CYP 3A4 could be detected in C3A-Unoptimized cells but was undetectable in non-transfected C3A cells. Im- munohistochemical staining indicated higher expression of GSTA1 in C3A-Unoptimized cells than in non-transfected C3A cells. The ability to metabolize lidocaine for C3A-Unoptimized cells was enhanced compared to non-transfected C3A cells （62.5% vs 30%）. CONCLUSION： The function of the C3A-Unoptimized cell line has been improved, and this cell line might be used in the bioartificial liver support system.

关 键 词：细胞色素P4503A4 谷胱甘肽硫转移酶A1 药物代谢 生物人工肝支持系统 

分 类 号：R735.7[医药卫生—肿瘤]