中国汉族人群24种CYP2C19新突变体的体外活性研究  

In vitro functional characterization of 24 newly-found CYP2C19 mutants in Chinese Han population

作　　者：李传保[1] 戴大鹏[2] 蔡杰[3] 耿培武[4] 王双虎[4] 王豪[3] 胡国新[3] 蔡剑平[2] 

机构地区：[1]卫生部北京医院检验科,北京100730 [2]卫生部北京医院,卫生部北京老年医学研究所 [3]温州医科大学药学院药理教研室 [4]浙江省丽水市人民医院临床药学实验室

出　　处：《山西医科大学学报》2015年第5期428-433,共6页Journal of Shanxi Medical University

基　　金：国家自然科学基金资助项目(31371280);卫生部行业科研专项经费资助项目(201302008)

摘　　要：目的以S-美芬妥英为探针药,体外分析24种汉族人群CYP2C19新变异体的酶学活性。方法以野生型CYP2C19 c DNA为模板,通过定点诱变方法获得24种CYP2C19新变异体和典型变异体CYP2C19.3的c DNA,利用Bac-toBac Baculovirus Expression System试剂盒,获得高表达各型CYP2C19变异体的昆虫微粒体。Western blot法进行CYP2C19蛋白定量。以S-美芬妥英为探针底物药,体外测定不同药物浓度下各型CYP2C19变异体的最大反应速率Vmax和米氏常数Km,评价新突变类型对CYP2C19体外代谢活性的影响。结果利用昆虫表达系统,成功获得高表达典型变异体CYP2C19.3和24种CYP2C19新变异体的昆虫微粒体。Western blot结果显示,变异体CYP2C19.3和35FS无正常CYP2C19酶的表达,与野生型相比,变异体CYP2C19.29和T130M表达量明显下降,其余变异体蛋白表达量与野生型接近。体外代谢实验结果显示,变异体L16F针对S-美芬妥英的体外清除率(Vmax/Km)较野生型升高,T130M的活性与野生型相似,其余各突变型的清除率均低于野生型。其中,突变体CYP2C19.3、35FS和R124Q对于该底物药无代谢活性。结论新发现的24种CYP2C19基因变异大多可引起酶体外药代活性显著降低,提示携带此类基因突变的个体服用经由CYP2C19代谢药物时,体内药物代谢速度可能会显著变慢,临床用药时需密切关注。Objective To investigate the functional characteristics of 24 novel CYP2C19 variants in Han Chinese population. Methods Full-length c DNA fragments of CYP2C19. 3 and 24 newly-found variants of CYP2C19 were obtained by polymerase chain reaction（PCR） site-directed mutagenesis using wild type CYP2C19. 1 as the template. According to the manufacturer＇s instruction,the insect cell microsomes highly expressing CYP2C19. 3 and 24 CYP2C19 variants were obtained with Bac-to-Bac Baculovirus Expression System.Then CYP2C19 protein expression level for each variant was quantified by Western blot. S-mephenytoin was used as a probing substrate for assessing the in vitro metabolic characteristics of each CYP2C19 variant. Vmaxand Kmvalues were calculated and used for evaluating the biological function of each mutant. Results Insect cell microsomes highly expressing 24 CYP2C19 variants were obtained successfully. The results of Western blot revealed that no CYP2C19 was expressed in microsomes carrying the variants CYP2C19. 3 and 35 FS.Compared with wild type,the expression of CYP2C19 was decreased significantly in microsomes carrying the variants CYP2C19. 29 and T130 M. The expression quantity of other mutants was close to that of wild type. The in vitro metabolism experimental results showed that the clearance rate（ Vmax/ Km）of variant L16 F towards S-mephenytoin was higher than that of wild type,whereas the variant T130 M exhibited similar clearance rate to that of wild type. The clearance rates of other variants were lower than that of wild type. Specifically,variants CYP2C19. 3,35 FS and R124 Q didn＇t show any metabolic activity to S-mephenytoin. Conclusion Most of the 24 novel variants of CYP2C19 greatly reduce the in vitro enzymatic activity of CYP2C19. While taking the drugs metabolized by CYP2C19,the patients carrying this mutated allele should take more cautions because their metabolic rates are slower than those of wild-type carriers.

关 键 词：CYP2C19 突变体 体外研究 

分 类 号：R394[医药卫生—医学遗传学]