摘要
不同个体对相同药物的应答不尽相同,相同药物也可能对不同个体产生不同的毒性,药物反应的个体差异与个体间药物代谢动力学的差异密切相关,而参与药物代谢过程的药物代谢酶是决定药物代谢动力学性质的关键因素。其中,细胞色素P4503A4(CYP3A4)是一类重要的药物代谢酶,约占肝脏及肠道中细胞色素P450酶(CytochromeP450, CYP)总含量的50%以上,其转录调控机制比较复杂,具有基因多态性和药物代谢催化活性多态性。个体间肝脏酶表达量约有40倍差异,在药物代谢能力方面也达到10倍左右差异。目前已公布的CYP3A4的等位基因突变,其分布频率较低,且存在种族差异。所以,基因多态性不能完全解释CYP3A4药物代谢的多态性,性别、外源药物、内源性物质、环境化学物质等都会引起CYP3A4药物代谢的差异。本文主要对CYP3A4的mRNA表达,蛋白质表达及代谢硝苯地平的酶活性在中国人群的分布情况进行研究,旨在探明CYP3A4药物代谢催化活性多态性受转录和翻译调控的程度及三个表型数据在人群的分布情况。
共收集了45例中国人肝组织样本,制备人肝微粒体,提取总RNA；实时定量PCR检测CYP3A4mRNA表达水平,蛋白质印迹法(Western blot)检测CYP3A4蛋白的表达水平,以硝苯地平为底物,用高效液相色谱法(HPLC)检测CYP3A4的酶活性；首先使用GraphPad Prism 5.0软件中的Shapiro-Wilk正态检验法检验这三个表型数据中国人群的分布情况是否符合正态分布,然后Spearman相关性检验分析这些表型之间的相关性,并使用Mann-Whitney检验法对样本供体的性别、年龄、病理状态对表型数据的影响进行分析。
45例肝组织CYP3A4mRNA表达水平存在巨大的个体差异,最大值与最小值之比为420,且mRNA表达不服从正态分布(p<0.0001)；CYP3A4蛋白表达水平差异达52倍,不符合正态分布(p<0.001)；CYP3A4对硝苯地平的代谢活性的最大值(2843pmol/min/mg)是最小值(121.7pmol/min/mg)的23倍,整体数据符合正态分布(p为0.03)。相关性检验结果显示CYP3A4mRNA表达水平与蛋白质表达水平显著相关(相关系数rs为0.5802,p<0.0001),CYP3A4mRNA表达水平与氧化硝苯地平的生成速率也显著相关(rs=0.5157,p<0.001),CYP3A4蛋白质表达水平与氧化硝苯地平生成速率高度相关(rs=0.7650,p<0.0001)。由此说明CYP3A4酶活性的变化与其mRNA和蛋白表达水平相关,但与受转录调控的程度相比,转录后和蛋白翻译水平的调控起了更大作用。另外本文的研究结果显示男性和女性的氧化硝苯地平生成速率、CYP3A4mRNA和蛋白表达水平没有显著性差异,且供体的年龄与氧化硝苯地平的生成速率、CYP3A4mRNA和蛋白表达水平均没有相关性,供体的病理状态的表型数据间的比较也没有得出显著性的差异。
总之,本研究发现中国人肝组织CYP3A4酶活性存在着明显的个体差异,这种差异与mRNA.蛋白表达水平相关。由于CYP3A4参与众多药物的代谢,因此本研究得到的信息对于未来进一步探究造成CYP3A4药物代谢差异的分子机制,指导经CYP3A4代谢的药物的个体化用药具有重要的意义。
Individuals may have different responses and may show different toxic effects to the same drug, which is closely correlated with interindividual variability in drug metabolism. Drug metabolizing enzymes is the important factor in drug metabolism. CYP3A4, which accounts for half of the content in human liver and intestinal CYPs, is a kind of important drug metabolizing enzymes. The mechanism in gene expression regulation of CYP3A4 is complex. Gene polymorphism and phenotypic polymorphism are also found in CYP3A4. It has been repoted that there was 40 times variability in the empression of liver enzymes with different people, where 10 times variability in drug metabolism. The frequency of the mutant alleles that has been reported is low and the distribution of the mutant alletes among different ethnic is variability. The impact of a polymorphism depends not only upon the gene polymorphism but also on other factor, such as the gender, age, other drugs, endogenous substances and environment. In this report, we examined the population distribution of CYP3A4 mRNA, protein and enzyme activity in Chinese people and determined whether the variability in CYP3A4 metabolic function was exhibited at both transcription and translation levels.
45 cases of human liver microsomes were prepared. RNA was extracted from human liver tissues. We measured levels of CYP3A4 mRNA by quantitative real-time PCR, protein quantification was performed by western blotting and CYP3A4 activity was measured by analysis of the rate of nifedipine oxidization with HPLC. The distribution and correlation of these determinations was analyzed using GraphPad Prism 5.0 software. At first whether the distribution is a normal one was analyzed using Shapiro-Wilk normality test. Relationships between each of the phenotype parameters(activities, relative protein level and relative mRNA level)were examined by Spearman correlation analysis. The effect of donor gender, age and pathological condition on each of the phenotype parameters was evaluated by the Mann-Whitney rank sum test.
A 420-fold variation of CYP3A4 mRNA expression was shown, and the CYP3A4 mRNA expression was non normally distributed (p<0.0001), coefficient of variation(CV)(104.11%). As for protein expression and activity, it was a 52-fold and 23-fold respectively. The distribution of protein expression did not follow a normal distribution (p<0.001), CV (47.61%). While the protein activity was unimodal distribution with normally distributed. CV(41.11%). The correlation coefficient between CYP3A4mRNA lever and protein level is 0.5802,p In conclusion, there is interindividual variability in CYP3A4 metabolic fuction which is correlated with the epression ofmRNA and protein. CYP3A4 is involved in numerous drug metabolism. Therefore, information obtained in this study is of significance to further explore differences in CYP3A4 drug metabolism caused by the molecular mechanisms and to guide individualized medicine which metobolized by CYP3A4.
引文
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