CYP3A5基因型的差异对环孢素药代动力学影响的体外研究

目的体外明确CYP3A5基因多态性对CYP3A5蛋白酶表达量的影响;不同的CYP3A5基因型对环孢素体外代谢的影响。方法采用PCR-RFLP方法和测序分析了21份肝脏标本的CYP3A5*3基因型;Western-blot方法对蛋白质进行定量;将CYP3A4特异性抑制剂酮康唑和环孢素一起孵育后,采用LC/MS-MS检测AM9/CsA浓度。结果CYP3A5*1/*1、CYP3A5*1/*3基因型和CYP3A5*3/*3基因型样本的蛋白表达量分别为:260.10±136.89nmol.g-1,85.44±55.22nmol.g-1和19.88±17.33nmol.g-1。各基因型蛋白表达量存在差异的同时,也存在一定程度的性别差异。采用AM9/CsA来评估CYP3A5活性,人肝微粒体孵育环孢素,CYP3A5*1/*1的活性明显强于CYP3A5*3/*3;量化校正CYP3A5后,此活性不存在性别差异。结论CYP3A5基因多态性可引起蛋白酶表达量的差异,因而对携带有CYP3A5*3个体,应用比CYP3A5*1/*1个体更小剂量的环孢素就能达到所需要的治疗血药浓度。

Effects of CYP3A5 genotype on CsA pharmacokinetics variances in vitro

Aim To identify the association between CYP3A5 genotype and CYP3A5 protein and to evaluatethe influence the CYP3A5 gene polymorphisms on CsA metabolism in vitro.Methods Through polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and sequencing assays,the allelic frequency of the CYP3A53 of 21 liver samples was determined.CYP3A5 protein quantity was determined by western-blot;After liver microsomes incubation with CsA and ketoconazole,the AM9 and CsA concentration was detected by LC/MS-MS.Results The protein conceutration of CYP3A51/1、CYP3A51/3 and CYP3A53/3 genotype were(260.10 ±136.89) nmol·g-1,85.44 ± 55.22 nmol·g-1 and 19.88 ± 17.33 nmol·g-1,respectively.Different genotype had different protein expression,but there was not stadistical difference between sexeven in the same genotype.CsA incubation in vitro suggested the enzyme activity of HLM from CYP3A51/1 genotype sample was much higher than that from CYP3A53 genotype sample when using AM9/CsA as the appraisal index.Conclusion The polymorphisms of CYP3A5 might induce the quantity difference of CYP3A5 protein.Compared with CYP3A51/1 individual,ones with CYP3A53 allele would need a fewer dosage of CsA to obtain the target concentration.