稳定表达人细胞色素P4501A2的HepG2细胞的建立及其代谢效应

Establishment of a HepG2 cell line stably expressing human cytochrome P450 1A2 and its metabolic activity

目的 :建立稳定表达人细胞的色素 P4 5 0 1A2 (CYP1A2 )的 Hep G2细胞。方法 :将所克隆的野生型CYP1A2 c DNA从重组质粒 p GEM- CYP1A2中用 Kpn / Bam H 双酶切 ,并亚克隆到哺乳动物细胞表达载体p REP9中。再将重组质粒转化感受态大肠杆菌 Top10 ,用氨苄青霉素抗性筛选和限制酶谱鉴定。改良的磷酸钙介导的细胞转染法将重组质粒 p REP9- CYP1A2转染肝癌细胞 Hep G2 ,用 RT- PCR技术对转基因细胞的 CYP1A2m RNA表达作了分析 ,并用 MTT法比较转基因细胞对黄曲霉素 B1(AFB1)细胞毒敏感试验。结果 :与 Hep G2细胞相比 ,Hep G2 - CYP1A2转基因细胞表达 CYP1A2 m RNA,能增强 AFB1的细胞毒作用。结论 :建立了稳定表达CYP1A2的转基因细胞系 ,可用于由 CYP1A2参与的毒理学与药物代谢研究。

Objective: To establish a HepG2 cell line stably expressing the human cytochrome P450 1A2 and to study its metablic activity. Methods: The human wild-type CYP1A2 cDNA was subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP1A2 to HepG2 cells. The expression of CYP1A2 mRNA was validated by RT-PCR. The metabolic activation of HepG2-CYP1A2 cells on aflatoxin B1 (AFB1) was assayed by cytotoxicity test. Results: The HepG2-CYP1A2 cells expressed CYP1A2 mRNA and could increase the cytotoxicity to AFB1 in comparison with that of wild-type HepG2 cells. Conclusion: The established HepG2-CYP1A2 can express the mRNA and has the metabolic activity to AFB1. The cell line may be useful for testing the toxicity and metabolism of xenobiotics, which might possibly be activated or metabolized by CYP1A2.