Summary： In adult liver, CYP3A4 plays an important role in the metabolism of a wide range of en- dogenous and exogenous compounds. To investigate whether there is a single nucleotide polymorphism （SNP） of CYP3A4 in...Summary： In adult liver, CYP3A4 plays an important role in the metabolism of a wide range of en- dogenous and exogenous compounds. To investigate whether there is a single nucleotide polymorphism （SNP） of CYP3A4 intron 2 in the liver and its effects on the mRNA expression and enzymatic activity of CYP3A4, genomic DNA was extracted from 96 liver tissue samples obtained from patients who had undergone liver surgery. An SNP of CYP3A4 intron 2 was identified by polymerase chain reaction （PCR）-single-strand confirmation polymorphism and DNA sequencing. The mRNA expression of CYP3A4 was determined by the fluorescence quantitative PCR technique. The enzymatic activity of CYP3A4 was measured using erythromycin and testosterone as probe substrates. Twelve patients were found to have the SNP/T4127G CYP3A4 within intron 2. The mRNA levels of CYP3A4 in wild-type and SNP/T4127G samples were 2.62±1.09 and 2.79±1.63, respectively （P〉0.05）. Erythromycin N-demethylase activity in wild-type and SNP/T4127G samples were 121.2±32.8 and 124.7±61.6 nmol·mg^-1min^-1, respectively （P〉0.05）. The activity of testosterone 613-hydroxylase was significantly different between wild-type （648±173 pmol·mg^-1·min^-1） and SNP/T4127G samples （540-4-196 pmol.mg-l-minl; P〈0.05）. In conclusion, the SNP/T4127G of CYP3A4 intron 2 exists in the liver. This SNP does not affect the mRNA expression of CYP3A4 but significantly decreases the hepatic micro- somal testosterone 613-hydroxylase activity of CYP3A4. Furthermore, this study indicates that the ap- propriate selection of probe substrates is very important in studying the relationship between the geno- type and phenotype of CYP3A4.展开更多
目的:探讨茵栀黄对CYP3A4转录表达的影响及机制。方法:Chang Liver细胞中加入0.01、0.1和1μM茵栀黄分别作用24h、36h和48h,采用RT-PCR法和报告基因方法检测CYP3A4 m RNA的表达水平。结果:不同浓度茵栀黄分别处理Chang Liver细胞24h和36...目的:探讨茵栀黄对CYP3A4转录表达的影响及机制。方法:Chang Liver细胞中加入0.01、0.1和1μM茵栀黄分别作用24h、36h和48h,采用RT-PCR法和报告基因方法检测CYP3A4 m RNA的表达水平。结果:不同浓度茵栀黄分别处理Chang Liver细胞24h和36h可明显升高CYP3A4 m RNA的表达水平(P<0.05);转入CAR载体的Chang Liver细胞经不同浓度茵栀黄处理36h均可激活CYP3A4的转录表达。结论:茵栀黄可能通过CAR介导增加Chang Liver细胞CYP3A4的转录表达。展开更多
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...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.展开更多
基金supported by grants from the Scientific Research Foundation for Returned Scholars of the Ministry of Education of ChinaPre-research Foundation of Wuhan University,China(No.301270050)
文摘Summary： In adult liver, CYP3A4 plays an important role in the metabolism of a wide range of en- dogenous and exogenous compounds. To investigate whether there is a single nucleotide polymorphism （SNP） of CYP3A4 intron 2 in the liver and its effects on the mRNA expression and enzymatic activity of CYP3A4, genomic DNA was extracted from 96 liver tissue samples obtained from patients who had undergone liver surgery. An SNP of CYP3A4 intron 2 was identified by polymerase chain reaction （PCR）-single-strand confirmation polymorphism and DNA sequencing. The mRNA expression of CYP3A4 was determined by the fluorescence quantitative PCR technique. The enzymatic activity of CYP3A4 was measured using erythromycin and testosterone as probe substrates. Twelve patients were found to have the SNP/T4127G CYP3A4 within intron 2. The mRNA levels of CYP3A4 in wild-type and SNP/T4127G samples were 2.62±1.09 and 2.79±1.63, respectively （P〉0.05）. Erythromycin N-demethylase activity in wild-type and SNP/T4127G samples were 121.2±32.8 and 124.7±61.6 nmol·mg^-1min^-1, respectively （P〉0.05）. The activity of testosterone 613-hydroxylase was significantly different between wild-type （648±173 pmol·mg^-1·min^-1） and SNP/T4127G samples （540-4-196 pmol.mg-l-minl; P〈0.05）. In conclusion, the SNP/T4127G of CYP3A4 intron 2 exists in the liver. This SNP does not affect the mRNA expression of CYP3A4 but significantly decreases the hepatic micro- somal testosterone 613-hydroxylase activity of CYP3A4. Furthermore, this study indicates that the ap- propriate selection of probe substrates is very important in studying the relationship between the geno- type and phenotype of CYP3A4.
文摘目的:探讨茵栀黄对CYP3A4转录表达的影响及机制。方法:Chang Liver细胞中加入0.01、0.1和1μM茵栀黄分别作用24h、36h和48h,采用RT-PCR法和报告基因方法检测CYP3A4 m RNA的表达水平。结果:不同浓度茵栀黄分别处理Chang Liver细胞24h和36h可明显升高CYP3A4 m RNA的表达水平(P<0.05);转入CAR载体的Chang Liver细胞经不同浓度茵栀黄处理36h均可激活CYP3A4的转录表达。结论:茵栀黄可能通过CAR介导增加Chang Liver细胞CYP3A4的转录表达。
文摘目的探讨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.
