本文選題：CYP2A6 + CYP2C9��； 參考：《鄭州大學(xué)》2014年碩士論文

【摘要】：細胞色素P450酶(cytochrome P450,CYP)又稱混合功能氧化酶和單加氧酶,是一類以血紅素為輔基的b族細胞色素的超家族蛋白酶,其中CYP2A6和CYP2C9不僅分別參與臨床約3%和15%藥物的代謝,還能代謝和/或活化多種前致癌物、前毒物及致突變劑。 目前發(fā)現(xiàn)多種藥物代謝酶含量和活性在個體間和種族間都呈現(xiàn)出顯著的差異性,從而在對臨床用藥、環(huán)境化合物和前致癌物的毒性作用的敏感性上表現(xiàn)出個體差異。通常CYP酶的基因多態(tài)性可能是引起個體差異的主要原因之一。 CYP2A6和CYP2C9基因具有高度的遺傳多態(tài)性,以往的相關(guān)研究多在重組酶體系和人體體內(nèi)展開,前者雖能較好的反映單一酶活性,但與人體內(nèi)環(huán)境相差較遠,后者雖接近人體內(nèi)環(huán)境,但涉及藥物體內(nèi)處置的多個環(huán)節(jié),不能單一反映代謝過程。綜合來說,人肝是人體最為接近的體外代謝平臺。 本研究擬以肝功能正常的人肝標(biāo)本為研究對象,考察基因多態(tài)性對CYP2A6和CYP2C9代謝探針?biāo)幬锵愣顾?coumarin, COH)和甲苯磺丁脲(tolbutamide,TOB)的影響,以期為臨床合理應(yīng)用經(jīng)CYP2A6和CYP2C9代謝的藥物提供理論依據(jù)。 方法 1人肝標(biāo)本 收集正常人肝標(biāo)本108例,年齡20-75歲(中位數(shù)為47歲)。男性和女性分別為36例和72例,有吸煙史和飲酒史的分別為12例和96例,用藥史顯示近期均未用明顯影響CYP酶活性的藥物。本試驗方案經(jīng)鄭州大學(xué)醫(yī)學(xué)倫理委員會批準(zhǔn),受試者簽署知情同意書。 2CYP2A6和CYP2C9的基因分型 采用DNA提取試劑盒從肝組織中提取基因組DNA。用CYP2A6、CYP2C9引物擴增含突變位點的基因片段,通過測序確定受試者的基因型。 3人肝微粒體的制備 采用差速離心法制備人肝微粒體,Bradford法測定肝微粒體蛋白含量。CYP2A6和CYP2C9的體外探針分別選擇香豆素和甲苯磺丁脲,并以探針?biāo)幬锏纳镛D(zhuǎn)化程度(TR)作為反應(yīng)速度。 4探針?biāo)幬锛按x物濃度的測定 人肝微粒體孵育體系中加入探針?biāo)幬�、磷酸鹽緩沖液等,預(yù)孵育后加入NADPH啟動反應(yīng),孵育后加入高氯酸終止反應(yīng),渦旋、離心,取上清進行分析測定。并進行孵育條件的優(yōu)化,選擇最合適的孵育時間和微粒體蛋白濃度。實驗所用探針?biāo)幬锛捌浞跤w系中代謝物的濃度測定均選用HPLC法。結(jié)果表明,所建立的藥物測定方法專屬性高,精密度、回收率良好,RSD15%,均符合生物樣本的測定要求。 5人肝酶動力學(xué)參數(shù)的測定 CYP2A6底物香豆素的濃度范圍為0.15625～20μM,CYP2C9底物甲苯磺丁脲的濃度范圍為31.25～2000μM。CYP2A6和CYP2C9孵育體系中蛋白濃度分別為0.3mg·ml-1和0.5mg·ml-1孵育時間分別為30min和60min,分別測定105例人肝CYP2A6和108例人肝CYP2C9的酶動力學(xué)參數(shù)Km、Vmax和CLint。 6統(tǒng)計方法 采用GraphPad Prism5.0軟件計算酶動力學(xué)參數(shù),SPSS17.0統(tǒng)計軟件對各項數(shù)據(jù)資料進行統(tǒng)計學(xué)分析。酶動力學(xué)參數(shù)進行正態(tài)性檢驗,對于非正態(tài)分布的,采取非參數(shù)檢驗進行相關(guān)分析。檢驗水準(zhǔn)α為0.05,P0.05認為有統(tǒng)計學(xué)意義。 結(jié)果 1CYP2A6基因型對人肝微粒體代謝香豆素的影響 1.1人肝微粒體代謝香豆素的酶動力學(xué) 正態(tài)性檢驗結(jié)果表明,人肝微粒體CYP2A6代謝香豆素的酶動力參數(shù)呈非正態(tài)分布,因此本實驗采用非參數(shù)檢驗進行統(tǒng)計分析。105例人肝微粒體代謝香豆素的酶動力學(xué)參數(shù)K-m、Vmax和CLint分別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差異倍數(shù)分別為12.9、386.5和453.9倍。結(jié)果顯示,CYP2A6代謝香豆素的酶動力學(xué)參數(shù)Km、Vmax和CLint呈現(xiàn)出較大的個體差異性。 1.2CYP2A6*1B基因型與香豆素代謝 CYP2A6*1B的突變頻率為53.3%。CYP2A6代謝香豆素的酶動力學(xué)參數(shù)Km、Vmax和CLint在CYP2A6*1A/*1A,*1A/*1B和*1B/*1B三組中都無顯著的差異性。結(jié)果顯示,CYP2A6*1B突變對人肝CYP2A6的代謝活性無明顯影響。 1.3CYP2A6*4基因型與香豆素代謝 105例樣本中基因型為CYP2A6*1/*和*1/*4的樣本分別有90和15例。CYP2A6*4的突變頻率為7.1%CYP2A6*1/*1和*1/*4基因型人肝微粒體Km、*max和CLint分別為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均顯著高于后者(P0.05)。結(jié)果顯示,CYP2A6*4突變明顯降低人肝CYP2A6對香豆素的代謝活性。 1.4CYP2A6*9基因型與香豆素代謝 CYP2A6*9的突變頻率為23.3%。CYP2A6*1/*1、*1/*9和*9/*9基因型人肝微粒體的Km分別為2.72(0.98～10.09)、2.09(0.87～6.18)和1.27(0.89～1.96)μM,前者顯著高于后兩者(P0.05)。CYP2A6*9/*基因型的Vmax為179.2(117.4～248.9)pmol-min-1·mg-1protein顯著低于*1/*1基因型417.2(49.7～1430.0)pmol-min-1·mg-1protein。結(jié)果顯示,CYP2A6*9突變明顯降低CYP2A6對香豆素的代謝活性。 2CYP2C9基因型對人肝微粒體代謝甲苯磺丁脲的影響 2.1人肝微粒體代謝甲苯磺丁脲的酶動力學(xué) 正態(tài)性檢驗結(jié)果表明,人肝微粒體CYP2C9代謝甲苯磺丁脲的酶動力參數(shù)呈非正態(tài)分布,因此本實驗采用非參數(shù)檢驗進行統(tǒng)計分析。108例人肝微粒體代謝甲苯磺丁脲的酶動力學(xué)參數(shù)Km、Vmax和CLint分別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差異倍數(shù)分別為5.5、5.5和24.6倍。結(jié)果顯示,CYP2C9代謝甲苯磺丁脲的酶動力學(xué)參數(shù)Km、Vmax和CLint呈現(xiàn)出較大的個體差異性。 2.2CYP2C9*3基因型與甲苯磺丁脲代謝 108例樣本中基因型為CYP2C9*1/*1、*1/*3的樣本分別有102和8例。CYP2C9*3的突變頻率為2.78%。CYP2C9*1/*1、*1/*3基因型人肝微粒體的Km、Vmax和CLint分別為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顯著低于后者,Vmax和CLint均顯著高于后者(P0.05)。結(jié)果顯示,CYP2C9*3突變明顯降低人肝CYP2C9對甲苯磺丁脲的代謝活性。 結(jié)論 1.人肝CYP2A6和CYP2C9對其藥物代謝呈現(xiàn)出了較大的個體差異性。 2. CYP2A6*4、CYP2A6*9突變明顯降低人肝微粒體CYP2A6的代謝活性；而CYP2A6突變對其代謝活性則無明顯影響。 3. CYP2C9*3突變明顯降低人肝微粒體CYP2C9的代謝活性。 4.人肝CYP2A6和CYP2CP相同基因型內(nèi)部代謝活性仍有較大差異。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R969.1

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本文編號：1958345