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目的体外研究尿苷二磷酸葡萄糖醛酸转移酶(UGT2B7)基因单核苷酸多态性(SNP)rs7439366对霉酚酸酯(MMF)代谢的影响。方法采用基因重组、定点突变技术构建UGT2B7基因rs7439366位点不同等位基因的过表达载体,Lipo2000转染法将重组载体转入HEK293细胞,采用LC/MS/MS系统检测霉酚酸(MPA)代谢产物7-O-葡醛酸苷(MPAG)24h的生成量来评估转染不同等位基因细胞的酶活性。结果成功构建p IRES2-EGFP-prom(T)和p IRES2-EGFP-prom(C)重组过表达载体并转染至HEK293细胞。LC/MS/MS系统检测结果显示携带C等位基因的突变型UGT2B7转化MPA的酶活性降低,24h产物MPAG的生成量为野生型的63.3%(P<0.001)。结论 UGT2B7基因SNP rs7439366可影响对MMF的代谢能力,是导致患者间MMF代谢差异的因素之一。
Objective To study the effect of single nucleotide polymorphism (SNP) rs7439366 of uridine diphosphate glucuronosyltransferase (UGT2B7) gene on the metabolism of mycophenolate mofetil (MMF) in vitro. Methods The overexpression vector of UGT2B7 rs7439366 locus was constructed by gene recombination and site-directed mutagenesis. The recombinant vector was transfected into HEK293 cells by lipofectamine 2000, and the mycophenolic acid (MPA) metabolism was detected by LC / MS / MS. Product 7-O-glucuronide (MPAG) for 24h to assess enzyme activity in cells transfected with different alleles. Results The pESES2-EGFP-prom (T) and pIRES2-EGFP-prom (C) recombinant overexpression vectors were successfully constructed and transfected into HEK293 cells. The results of LC / MS / MS showed that the enzymatic activity of mutant UGT2B7 carrying C allele was lower than that of MPA. The amount of MPAG produced in 24h was 63.3% of the wild type (P <0.001). Conclusion UGT2B7 gene SNP rs7439366 can affect the metabolism of MMF, which is one of the factors leading to differences in MMF metabolism among patients.