摘要
目的:体外研究尿苷二磷酸葡萄糖醛酸转移酶(UGT2B7)基因SNP rs28365062对霉酚酸酯(MMF)代谢的影响,明确该位点变异是否与MMF副作用具有潜在关系。方法:采用基因重组、定点突变技术构建UGT2B7基因SNP rs28365062不同等位基因过表达载体,POLO3000转染法将重组过表达载体转染HEK293细胞,采用液相色谱-质谱联用仪(LC/MS/MS)系统检测霉酚酸(MPA)代谢产物--酰基化葡萄糖醛酸化物(Ac MPAG)24小时的生成量来评估转染不同等位基因细胞的酶活性。结果:成功构建过表达载体p IRES2-EGFP-prom(A)和p IRES2-EGFP-prom(G)并转染至HEK293细胞。LC/MS/MS系统检测结果显示携带G等位基因的突变型UGT2B7代谢MMF产生Ac MPAG的能力降低,产物Ac MPAG 24小时的生成量仅为野生型的18.60%(P<0.001)。结论:UGT2B7基因SNP rs28365062可显著影响MMF代谢产物Ac MPAG的生成,可能是导致病人对MMF产生不同程度副作用的因素之一。
Objective： To investigate the effect of A/G mutation in SNP rs28365062 site of uridine diphosphate glucuronosyltransferase（UGT2B7）on mycophenolate mofetil metabolism, clear the relationship between this mutation and side effect of MMF. Methods： Wild and mutant type overexpression vectors containing UGT2B7 gene were constructed by recombinant gene technique and the strategy of site-directed mutagenesis. Then, target genes were transfected into HEK293 cells by polo3000. LC/MS/MS system was used to detect the yields of Ac MPAG produced by different types of HEK293 cells in 24 hours, then, these cells transfected by different alleles will be assessed by their enzyme activity. Results： The overexpression vectors p IRES2-EGFP-prom（A）and p IRES2-EGFP-prom（G）were successfully constructed and transfected into HEK293 cells. The results analyzed by LC/MS/MS showed that the cells containing mutant type of UGT2B7 gene with G allele produced less Ac MPAG than that with A allele, generation amount in24 hours of Ac MPAG is 18.60 % of UGT2B7 wild type,（P〈0.001）. Conclusions： UGT2B7 gene SNP rs28365062 can significantly affect the production of Ac MPAG,which may be one of the factors leading to patients have different degrees of side effects to MMF.
出处
《现代生物医学进展》
CAS
2015年第30期5811-5815,共5页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(81270557
81110108010)
国家青年科学基金项目(81000188)
