人细胞色素P4502B6在哺乳类细胞中的稳定表达

Stable expression of human cytochrome P4502B6 in mammalian cell lines

〗在诱变试验中广泛应用人羊膜ＦＬ细胞，中国仓鼠ＣＨＬ和Ｖ７９细胞，但由于它们不能表达内源性细胞色素Ｐ４５０（ＣＹＰ）活化前致突变物，而严重限制了它们的使用．本工作构建了携有编码ＣＹＰ２Ｂ６单胺氧化酶的ｃＤＮＡ重组表达体并分别导入上述三种细胞中表达，经Ｇ４１８抗性筛选，建立了ＦＬ－２Ｂ６，ＣＨＬ－２Ｂ６和Ｖ７９－２Ｂ６三种转基因细胞系，用Ｎｏｒｔｈｅｒｎ印迹杂交证明它们都有ＣＹＰ２Ｂ６的表达．在ＣＨＬ－２Ｂ６细胞中还测得较高水平的７－乙氧基－４－三氟甲基香豆素去乙基酶活性，它对前致突变物甲基亚硝胺吡啶酮的致微核形成作用有很高敏感性，并呈剂量效应关系，但其母系细胞皆为阴性．

Human FL cell line, Chinese hamster CHL and V79 cell lines are widely used for mutagenicity testing. However, they can not express cytochrome P-450(CYP), a necessary enzyme for the oxidation of xenobiotics and activation of promutagens/procarcinogens and thus their applications are limited. A full-length cDNA clone encoding the monooxygenase CYP2B6 was constructed and introduced into FL, CHL and V79 cell lines. The new cell lines (FL-2B6, CHL-2B6 and V79-2B6) had been established after G418 selection. Northern blotting showed that all of them expressed CYP2B6 gene.7-Ethoxy-4-trifluoromethylcoumarin O -deethylation (EFCOD) activity and the bioactivation capacity for nitrosamine promutagen 4-methylnitrosamino- 1-(3-pyridyl)-1-butanone (NNK) were determined. In postmitochondrial supernatant and microsome from the CHL-2B6 cell, the EFCOD activity was 0.84±0.17 and 20.0±2.4 pmol·min -1 ·mg -1 protein respectively, whereas it could not be detected in the parent cell line. And, the CHL-2B6 cell line reproducibly displayed high sensitivity dose-dependently to NNK in inducing micronuclei, while its parent cell line could not.