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

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.

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

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.