【摘要】 目的探讨CYP3A4内含子10及其内SNP CYP3A4*1G对CYP3A4基因表达的调控作用.方法构建CYP3A4启动子启动转录的CYP3A4真核表达质粒,将CYP3A4内含子10(CYP3A4*1G野生型/突变型)正向或反向插入该质粒的启动子上游,从而构建出不同基因型、不同方向的CYP3A4内含子10调控的CYP3A4真核表达质粒.将构建好的各质粒分别转染入Hep G2细胞,应用实时荧光定量PCR方法检测CYP3A4 m RNA表达水平.结果构建的重组质粒经酶切验证与测序鉴定,插入片段的序列和方向与预期完全一致.无论CYP3A4*1G野生型还是突变型,反向组CYP3A4 m RNA表达水平均高于正向组(P<0.01);而CYP3A4内含子10无论是正向还是反向,CYP3A4*1G野生型组CYP3A4 m RNA表达水平均高于突变型组(P<0.01).此外,正向突变组CYP3A4 m RNA表达水平低于阳性对照组,不但没有增强CYP3A4启动子的转录作用反而有所减弱.结论CYP3A4内含子10有类似于增强子的功能,能够影响CYP3A4启动子对CYP3A4基因的转录,且具有方向性和CYP3A4*1G等位基因依赖性.更多还原

【Abstract】 Objective To explore the regulation of CYP3A4 gene expression by CYP3A4 intron 10 and CYP3A4*1G.Methods CYP3A4 eukaryotic expression vector which promoted by CYP3A4 promoter was constructed.The forward and reverse full-length segments of CYP3A4 intron 10(with CYP3A4*1G allele wild/mutant type) were ligated into the recombinant eukaryotic expression vector above at upstream of CYP3A4 promoter to construct CYP3A4 eukaryotic expression vector which promoted by CYP3A4 promoter and regulated by CYP3A4 intron 10 with different genotypes and directions.The vectors were transfected into Hep G2 respectively.The levels of CYP3A4 m RNA were detected by quantitative real-time PC.Results These insertion sequences and their directions were exactly correct.The expression level of CYP3A4 m RNA was higher when CYP3A4 intron 10 was inthe forward than in the reverse direction(P<0.01);the wild group showed higher CYP3A4 m RNA level than the mutant group(P<0.01);while the forward mutant group showed lower CYP3A4 m RNA level than the positive control group.Conclusion CYP3A4 intron 10 is like an enhancer functionally, which could influence the transcription of CYP3A4 promoter depending on its direction and regulation by CYP3A4*1G allele.更多还原