人肝微粒体CYP2A6和CYP2C9基因多态性对其药物代谢活性的影响

发布时间：2018-05-31 04:48

本文选题：CYP2A6 + CYP2C9　；参考：《郑州大学》2014年硕士论文

【摘要】：细胞色素P450酶(cytochrome P450,CYP)又称混合功能氧化酶和单加氧酶,是一类以血红素为辅基的b族细胞色素的超家族蛋白酶,其中CYP2A6和CYP2C9不仅分别参与临床约3%和15%药物的代谢,还能代谢和/或活化多种前致癌物、前毒物及致突变剂。目前发现多种药物代谢酶含量和活性在个体间和种族间都呈现出显著的差异性,从而在对临床用药、环境化合物和前致癌物的毒性作用的敏感性上表现出个体差异。通常CYP酶的基因多态性可能是引起个体差异的主要原因之一。 CYP2A6和CYP2C9基因具有高度的遗传多态性,以往的相关研究多在重组酶体系和人体体内展开,前者虽能较好的反映单一酶活性,但与人体内环境相差较远,后者虽接近人体内环境,但涉及药物体内处置的多个环节,不能单一反映代谢过程。综合来说,人肝是人体最为接近的体外代谢平台。本研究拟以肝功能正常的人肝标本为研究对象,考察基因多态性对CYP2A6和CYP2C9代谢探针药物香豆素(coumarin, COH)和甲苯磺丁脲(tolbutamide,TOB)的影响,以期为临床合理应用经CYP2A6和CYP2C9代谢的药物提供理论依据。方法 1人肝标本收集正常人肝标本108例,年龄20-75岁(中位数为47岁)。男性和女性分别为36例和72例,有吸烟史和饮酒史的分别为12例和96例,用药史显示近期均未用明显影响CYP酶活性的药物。本试验方案经郑州大学医学伦理委员会批准,受试者签署知情同意书。 2CYP2A6和CYP2C9的基因分型采用DNA提取试剂盒从肝组织中提取基因组DNA。用CYP2A6、CYP2C9引物扩增含突变位点的基因片段,通过测序确定受试者的基因型。 3人肝微粒体的制备采用差速离心法制备人肝微粒体,Bradford法测定肝微粒体蛋白含量。CYP2A6和CYP2C9的体外探针分别选择香豆素和甲苯磺丁脲,并以探针药物的生物转化程度(TR)作为反应速度。 4探针药物及代谢物浓度的测定人肝微粒体孵育体系中加入探针药物、磷酸盐缓冲液等,预孵育后加入NADPH启动反应,孵育后加入高氯酸终止反应,涡旋、离心,取上清进行分析测定。并进行孵育条件的优化,选择最合适的孵育时间和微粒体蛋白浓度。实验所用探针药物及其孵育体系中代谢物的浓度测定均选用HPLC法。结果表明,所建立的药物测定方法专属性高,精密度、回收率良好,RSD15%,均符合生物样本的测定要求。 5人肝酶动力学参数的测定 CYP2A6底物香豆素的浓度范围为0.15625～20μM,CYP2C9底物甲苯磺丁脲的浓度范围为31.25～2000μM。CYP2A6和CYP2C9孵育体系中蛋白浓度分别为0.3mg·ml-1和0.5mg·ml-1孵育时间分别为30min和60min,分别测定105例人肝CYP2A6和108例人肝CYP2C9的酶动力学参数Km、Vmax和CLint。 6统计方法采用GraphPad Prism5.0软件计算酶动力学参数,SPSS17.0统计软件对各项数据资料进行统计学分析。酶动力学参数进行正态性检验,对于非正态分布的,采取非参数检验进行相关分析。检验水准α为0.05,P0.05认为有统计学意义。结果 1CYP2A6基因型对人肝微粒体代谢香豆素的影响 1.1人肝微粒体代谢香豆素的酶动力学正态性检验结果表明,人肝微粒体CYP2A6代谢香豆素的酶动力参数呈非正态分布,因此本实验采用非参数检验进行统计分析。105例人肝微粒体代谢香豆素的酶动力学参数K-m、Vmax和CLint分别2.33(0.78～10.09)μM、354.4(3.7～1430.0) pmol·min-1·mg-1protein、144.5(1.2～544.7)μl·min-1·mg-1protein。 Km、Vmax和CLint差异倍数分别为12.9、386.5和453.9倍。结果显示,CYP2A6代谢香豆素的酶动力学参数Km、Vmax和CLint呈现出较大的个体差异性。 1.2CYP2A6*1B基因型与香豆素代谢 CYP2A6*1B的突变频率为53.3%。CYP2A6代谢香豆素的酶动力学参数Km、Vmax和CLint在CYP2A6*1A/*1A,*1A/*1B和*1B/*1B三组中都无显著的差异性。结果显示,CYP2A6*1B突变对人肝CYP2A6的代谢活性无明显影响。 1.3CYP2A6*4基因型与香豆素代谢 105例样本中基因型为CYP2A6*1/*和*1/*4的样本分别有90和15例。CYP2A6*4的突变频率为7.1%CYP2A6*1/*1和*1/*4基因型人肝微粒体Km、*max和CLint分别为2.44(0.87～10.09)和1.18(0.78～7.88)μM,374.5(49.7～1430.0)和42.5(3.7～495.1)pmol·min-1mg-1protein,147.4(27.4～544.7)和43.2(1.2～209.7)μl·min-1·mg-1protein,前者Km、Vmax和CLint均显著高于后者(P0.05)。结果显示,CYP2A6*4突变明显降低人肝CYP2A6对香豆素的代谢活性。 1.4CYP2A6*9基因型与香豆素代谢 CYP2A6*9的突变频率为23.3%。CYP2A6*1/*1、*1/*9和*9/*9基因型人肝微粒体的Km分别为2.72(0.98～10.09)、2.09(0.87～6.18)和1.27(0.89～1.96)μM,前者显著高于后两者(P0.05)。CYP2A6*9/*基因型的Vmax为179.2(117.4～248.9)pmol-min-1·mg-1protein显著低于*1/*1基因型417.2(49.7～1430.0)pmol-min-1·mg-1protein。结果显示,CYP2A6*9突变明显降低CYP2A6对香豆素的代谢活性。 2CYP2C9基因型对人肝微粒体代谢甲苯磺丁脲的影响 2.1人肝微粒体代谢甲苯磺丁脲的酶动力学正态性检验结果表明,人肝微粒体CYP2C9代谢甲苯磺丁脲的酶动力参数呈非正态分布,因此本实验采用非参数检验进行统计分析。108例人肝微粒体代谢甲苯磺丁脲的酶动力学参数Km、Vmax和CLint分别230.4(101.2～555.3)μM、254.1(82.4～454.8) pmol·min-1·mg-1protein、1.11(0.17～4.18)μl·min-1·mg-1protein。 Km、Vmax和CLint差异倍数分别为5.5、5.5和24.6倍。结果显示,CYP2C9代谢甲苯磺丁脲的酶动力学参数Km、Vmax和CLint呈现出较大的个体差异性。 2.2CYP2C9*3基因型与甲苯磺丁脲代谢 108例样本中基因型为CYP2C9*1/*1、*1/*3的样本分别有102和8例。CYP2C9*3的突变频率为2.78%。CYP2C9*1/*1、*1/*3基因型人肝微粒体的Km、Vmax和CLint分别为217.9(101.2～555.3)和324.7(266.3～510.9)μM,257.1(83.8-454.8)和172.6(82.4～207.0)pmol·min-1·mg-1protein,1.18(0.17～4.18)和0.47(0.30～0.78)μl·min-1·mg-1protein,前者Km显著低于后者,Vmax和CLint均显著高于后者(P0.05)。结果显示,CYP2C9*3突变明显降低人肝CYP2C9对甲苯磺丁脲的代谢活性。结论 1.人肝CYP2A6和CYP2C9对其药物代谢呈现出了较大的个体差异性。 2. CYP2A6*4、CYP2A6*9突变明显降低人肝微粒体CYP2A6的代谢活性；而CYP2A6突变对其代谢活性则无明显影响。 3. CYP2C9*3突变明显降低人肝微粒体CYP2C9的代谢活性。 4.人肝CYP2A6和CYP2CP相同基因型内部代谢活性仍有较大差异。
[Abstract]:The cytochrome P450 enzyme (cytochrome P450, CYP), also known as mixed function oxidase and monooxygenase, is a class of superfamily protease of B family cytochrome with heme, in which CYP2A6 and CYP2C9 are not only involved in the metabolism of clinical 3% and 15% drugs, but also metabolize and / or activate a variety of pre carcinogens, precursor and mutagens.
At present, there is a significant difference in the content and activity of various drug metabolizing enzymes between individuals and races, which shows individual differences in the sensitivity to the toxicity of clinical drugs, environmental compounds and pre carcinogens. The genetic polymorphism of CYP enzyme may be one of the main causes of individual differences.
CYP2A6 and CYP2C9 genes have high genetic polymorphism. The previous related studies are mostly in the recombinant enzyme system and human body. Although the former can better reflect the activity of the single enzyme, the difference is far from the human environment, although the latter is close to the internal environment, but many links involved in the treatment of drugs can not reflect the metabolic process alone. In general, human liver is the closest metabolic platform to human body.
In this study, the aim of this study was to investigate the effects of gene polymorphism on CYP2A6 and CYP2C9 metabolic probes, including coumarin (COH) and tolbutamide (tolbutamide, TOB), and to provide a theoretical basis for the rational use of CYP2A6 and CYP2C9 metabolism drugs.
Method
1 human liver specimens
108 cases of normal human liver specimens were collected, aged 20-75 years (median 47 years). Men and women were 36 and 72, respectively, with the history of smoking and alcohol consumption in 12 and 96 cases. The history of drug use showed that the drug was not significantly affected by CYP enzyme activity in the near future. The test program was approved by the medical ethics committee of Zhengzhou University, and the subjects signed the informed consent. Meaning book.
Genotyping of 2CYP2A6 and CYP2C9
The DNA extraction kit was used to extract genomic DNA. from the liver tissue, and CYP2A6, CYP2C9 primers were used to amplify the gene fragment containing the mutation site, and the genotypes of the subjects were determined by sequencing.
Preparation of 3 human liver microsomes
Human liver microsomes were prepared by differential centrifugation. Coumarin and tolulin were selected by Bradford method to determine the content of liver microsomal protein content.CYP2A6 and CYP2C9 respectively, and the bioconversion degree (TR) of the probe drug was used as the reaction rate.
Determination of the concentration of 4 probe drugs and metabolites
In the human liver microsomal incubation system, the probe drug, phosphate buffer solution, etc. are added to the incubation reaction, after incubation, the NADPH start reaction is added to the incubation system. After incubation, perchloric acid termination reaction, vortex, centrifuge, and supernatant are analyzed, and the incubation conditions are optimized to select the most suitable incubation time and the concentration of microsome protein. HPLC method was used to determine the concentration of metabolites in the incubating system. The results showed that the established method was highly exclusive, precision, recovery rate and RSD15%, which were in line with the requirements of biological samples.
Determination of the kinetic parameters of liver enzyme in 5 people
The concentration range of CYP2A6 substrate coumarin was 0.15625 ~ 20 mu M, and the concentration range of CYP2C9 substrate tolubutyurea was 31.25 ~ 2000 mu M.CYP2A6 and CYP2C9 incubated with 0.3mg. Ml-1 and 0.5mg. Ml-1 respectively for 30min and 60min. The enzyme kinetic parameters of 105 human liver CYP2A6 and 108 human hepatocytes were measured respectively. Km, Vmax and CLint.
6 statistical methods
The GraphPad Prism5.0 software was used to calculate the enzyme kinetic parameters, and the SPSS17.0 statistical software was used to analyze the data. The enzyme kinetic parameters were tested in normality, and the nonparametric test was used to analyze the non normal distribution. The test level was 0.05, and the P0.05 was statistically significant.
Result
Effects of 1CYP2A6 genotypes on metabolism of coumarin in human liver microsomes
Enzyme kinetics of microsome metabolism of coumarin in 1.1 human liver microsomes
The results of normal test showed that the enzyme dynamic parameters of CYP2A6 metabolite in human liver microsomes were nonnormal distribution. Therefore, the nonparametric test was used to analyze the enzyme kinetic parameters K-m, Vmax and CLint 2.33 (0.78 to 10.09) mu M, 354.4 (3.7 ~ 1430) pmol. Min-1. Mg-1prote, by nonparametric test. In, 144.5 (1.2 to 544.7) Mu L. Min-1. Mg-1protein. Km, Vmax and CLint were 12.9386.5 and 453.9 times respectively. The results showed that the enzyme kinetic parameters of CYP2A6 metabolism coumarin Km, Vmax and CLint showed a larger individual difference.
1.2CYP2A6*1B genotypes and coumarin metabolism
The mutation frequency of CYP2A6*1B was 53.3%.CYP2A6 metabolic coumarin's enzyme kinetic parameter Km, Vmax and CLint had no significant difference in CYP2A6*1A/*1A, *1A/*1B and *1B/*1B three groups. The results showed that the CYP2A6*1B mutation had no significant effect on the metabolic activity of human liver CYP2A6.
1.3CYP2A6*4 genotypes and coumarin metabolism
In the 105 samples, 90 and 15 cases of CYP2A6*1/* and *1/*4 were 7.1%CYP2A6*1/*1 and *1/*4 genotype of human liver microsomal Km, *max and CLint were 2.44 (0.87 to 10.09) and 1.18 (0.78 to 7.88), respectively, 374.5 (49.7 ~ 1430) and 42.5 (49.7 ~ 1430) and pmol min-1mg-1protein. 3.2 (1.2 to 209.7) Mu L. Min-1. Mg-1protein, the former Km, Vmax and CLint were significantly higher than those of the latter (P0.05). The results showed that CYP2A6*4 mutation significantly reduced the metabolic activity of CYP2A6 to coumarin in human liver.
1.4CYP2A6*9 genotypes and coumarin metabolism
The mutation frequency of CYP2A6*9 was 23.3%.CYP2A6*1/*1, and the Km of human liver microsomes in *1/*9 and *9/*9 genotype was 2.72 (0.98 to 10.09), 2.09 (0.87 to 6.18) and 1.27 (0.89 to 1.96) mu M, and the former was significantly higher than that of the latter (P0.05).CYP2A6*9/* genotype Vmax was 179.2 (117.4 ~ 248.9) pmol-min-1. Mg-1protein was significantly lower than *1/*1 genotype 417.2 (49.7). ~ 1430) pmol-min-1? Mg-1protein. results showed that CYP2A6*9 mutation significantly reduced the metabolic activity of CYP2A6 to coumarin.
Effects of 2CYP2C9 genotype on toluene urea metabolism in human liver microsomes
Enzymatic kinetics of toluene sulfate metabolism in 2.1 human liver microsomes
The results of normal test showed that the enzyme kinetic parameters of CYP2C9 metabolism of tolubutyurea in human liver microsomes were nonnormal distribution. Therefore, the nonparametric test was used to analyze the enzyme kinetic parameters Km, Vmax and CLint 230.4 (101.2 to 555.3) mu M, 254.1 (82.4 ~ 454.8) pmol. Min, respectively, by nonparametric test. -1. Mg-1protein, 1.11 (0.17 to 4.18) Mu L. Min-1. Mg-1protein. Km, Vmax and CLint are 5.5,5.5 and 24.6 times respectively. The results showed that the enzyme kinetics parameters of the CYP2C9 metabolize tolusulfonylurea were Km, Vmax and the individual showed a larger individual difference.
2.2CYP2C9*3 genotype and metabolism of tolubutyback
The genotype of 108 samples was CYP2C9*1/*1, 102 and 8 cases of.CYP2C9*3 were 2.78%.CYP2C9*1/*1, Km of *1/*3 genotype human liver microsomes, Vmax and CLint 217.9 (101.2 to 555.3) and 324.7 (266.3 ~ 510.9) micron respectively, 257.1 (83.8-454.8) and 172.6 (82.4 ~ 207) pmol, min-1. 8) and 0.47 (0.30 ~ 0.78) Mu L. Min-1. Mg-1protein, the former Km was significantly lower than the latter, Vmax and CLint were significantly higher than the latter (P0.05). The results showed that CYP2C9*3 mutation significantly reduced the metabolic activity of CYP2C9 to tolubutyback.
conclusion
The CYP2A6 and CYP2C9 of 1. human liver showed a great individual difference in drug metabolism.
2. CYP2A6*4, CYP2A6*9 mutation significantly reduced the metabolic activity of CYP2A6 in human liver microsomes, while CYP2A6 mutation had no significant effect on its metabolic activity.
3. CYP2C9*3 mutation significantly reduced the metabolic activity of CYP2C9 in human liver microsomes.
The metabolic activity of the same genotype of CYP2A6 and CYP2CP in 4. human liver is still quite different.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R969.1

【参考文献】