稳定表达细胞色素P4501A1人肝癌细胞系的建立

Establishment of human hepatoma cell line expressing cytochrome P4501A1

目的:建立稳定表达人细胞色素P4501A1(CYP1A1)的人肝癌细胞系(Bel-7402)。方法:通过将人细胞色素P4501A1(CYP1A1)的表达质粒转染到人肝癌细胞系(Bel-7402),经G418抗性筛选得到稳定的克隆并扩大培养成系,WesternBlotting检测蛋白的表达。细胞增殖试验鉴定CYP1A1的多环芳烃类底物二甲基苯并蒽(DMBA)对Bel-7402/1A1细胞的增殖抑制作用。双核微核实验对Bel-7402/1A1细胞进行了遗传毒理的短期实验。结果:转染后筛选得到的Bel-7402抗性克隆,Western Blotting检测表明有较高的CYP1A1蛋白表达。细胞暴露于DMBA时,Bel-7402/1A1与Bel-7402细胞相比,细胞增殖明显抑制。双核微核试验表明Bel-7402/1A1细胞的微核率随DMBA浓度增高而显著增加,Bel-7402无明显变化。连续传代后仍有CYP1A1标志酶EROD的较高表达,该酶活性能被CYP1A1抑制剂α-萘黄酮抑制。结论:建立了一个新的稳定表达CYP1A1的1个肝癌细胞系(Bel-7402/CYP1A1),可代谢多环芳烃化合物产生更大的毒性。该细胞系可用来筛选能被CYP1A1代谢的化合物,或能抑制CYP1A1活性的化合物,并可用来研究CYP1A1的催化性质及被CYP1A1代谢的化合物的毒性机理。

Aim：To establish a human hepatic carcinoma cell line （Bel-7402） which could express human cytochrome P4501Al（CYP1A1）. Methods： The recombinant plasmid containing CYP1A1 was stably transfected to Bel-7402 cells, and the cells were screened with G418 to obtain stable clone. Western Blotting was used to detect the expression of CYP1A1 protein. Moreorer, cell proliferation study was used to analyze whether Bel-7402/1A1 cells could metabolize the polycyclic aromatic hydrocarbons substrate of CYP1A1 （DMBA）. The short-term test of genetic toxicology was performed with micronucleus method. Results：Western blot showed that the transfected cells expressed CYP1A1 protein while untransfected cells not. When cells were exposed to DMBA, the proliferation of Bel-7402/1A1 cells was apparently inhibited compared with that of Bel-7402 cells. Micronucleus experiment indicated micronucleus frequence of Bel-7402/1A1 cells was significantly increased with increased substrate concentration, whereas Bel-7402 cells had no obvious transformation. The cells still had high EROD activity after successive subculture, and the enzyme activity could be inhibited partially by α-NF. Conclusion：These results indicated that a new hepatic carcinoma cell line （Bel-7402/CYP1A1） which expressed CYP1A1 protein has been established. This cell line could metabolize the substrate of CYP1AI（DMBA） and produce higher cytotoxicity. There fore, the cell line could be used as a helpful model to screen out the compounds which could be metabolized by CYP1A1 or inhibit the activity of CYP1A1 ,and be used in the research of catalytic character of CYP1A1 and mechamism of toxicology of compounds could be metablolized by CYP1A1.