构建 CYP2C8及其3种突变体细胞表达体系，以紫杉醇为底物研究 CYP2C8基因多态性对其酶活性的影响，以及构建 CYP2C8和 CYP3A4共转染细胞体系研究小分子激酶抑制剂对紫杉醇代谢途径的抑制。根据基因文库分别合成 CYP3A4以及CYP2C8及其3种突变体 CYP2C8*2（805A>T）、CYP2C8*3（416G>A，1196A>G）、CYP2C8*4（792C>G）的基因编码片段，将其连接到 PEGFP-N1表达质粒，测序验证。将 CYP2C8野生型及其突变体分别转染 HepG2细胞，24 h 后加入紫杉醇进行孵育，通过建立好的 LC-MS/MS 方法对代谢物进行定量分析。同时，也将野生型 CYP2C8和 CYP3A4质粒按一定的浓度比转入 HepG2细胞构建共表达体系。并筛选出合适的质粒浓度比转染细胞，在加入紫杉醇孵育时，同时加入小分子激酶抑制剂，考察小分子激酶抑制剂对紫杉醇代谢途径的抑制作用。结果表明，CYP2C8*4代谢酶对紫杉醇的代谢能力存在明显差异，其中 CYP2C8*2和 CYP2C8*3代谢活性分别是野生型的81%（P<0.05）和87%（P<0.05），而 CYP2C8*4则是野生型的65%（P<0.01）。尼洛替尼完全抑制了紫杉醇的代谢，阿法替尼对紫杉醇的两条代谢途径抑制达30%，而伊马替尼选择性抑制了 CYPD3A4的活性。不同基因型 CYP2C8对紫杉醇的代谢存在差异，可能是导致临床疗效不同的原因。小分子激酶抑制剂在与紫杉醇联合使用时，对紫杉醇代谢的抑制各不相同。%The aims of this work are to establish the cell expression system of CYP2C8 and its 3 mutants,to study the effects of gene polymorphisms on its enzymatic activities,and to investigate the effects of small molecular kinase inhibitors on paclitaxel metabolism with the established CYP2C8 and CYP3A4 co-transfected cell lines. The gene fragments of CYP3A4,CYP2C8 and its three mutants(CYP2C8*2 (805A>T),CYP2C8*3(416G>A,1196A>G),and CYP2C8*4(792C>G))were synthesized based on gene bank. Then those fragments were ligated to expression plasmid PEGFP-N1 for sequencing evaluation. Twenty-four hours after the wild and mutant plasmids were transfected into HepG2 cells,paclitaxel was added and incubation proceeded,and then the metabolites were quantitatively detected by well-constructed LC-MS/MS. Concurrently,the wild CYP2C8 and CYP3A4 plasmids in certain concentration ratios were transfected into HepG2 cells,to establish a co-expression system. Screening the plasmids with proper concentration ratio and transfecting them to the cells,adding small molecule kinase inhibitor while adding paclitaxel for incubation,the inhibition of paclitaxel metabolism by small molecule kinase inhibitors was investigated. The results showed that the metabolic enzyme of different CYP2C8 caused the significant difference of paclitaxel metabolic activity,of which the metabolic activity of CYP2C8*2,CYP2C8*3,CYP2C8*4 remained at about 80%(P < 0.05),87%(P < 0.05),and 65%(P < 0.01),respectively,as compared to the wild type. Nilotinib completely inhibited the metabolism of paclitaxel and axitinib showed a 30% inhibition,while imatinib didn’t have any inhibitory effect. As conclusion,different genotypes of CYP2C8 differentially affect the overall paclitaxel metabolism,which might be the reason that results in the varied treatment effect in clinic. Small molecule kinase inhibitors while combined use with paclitaxel may inhibit the metabolism of paclitaxel to different extents.
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