本文選題：CYP2D6 + PCR��； 參考：《中央民族大學(xué)》2012年碩士論文

【摘要】：[目的與意義] 藥物通常是和特定的靶點(diǎn)結(jié)合而產(chǎn)生藥物效應(yīng),因此藥物效應(yīng)與靶部位的藥物濃度有密切的關(guān)系,而靶部位的藥物濃度又與血藥濃度存在著動(dòng)態(tài)平衡。血藥濃度不僅取決于藥物劑量,而且還與各種藥物代謝動(dòng)力學(xué)參數(shù)密切相關(guān)。因此如能了解個(gè)體或群體的藥物代謝動(dòng)力學(xué)參數(shù),就能有效預(yù)測(cè)靶部位的藥物濃度,使個(gè)體化給藥成為可能。在各種藥物動(dòng)力學(xué)參數(shù)中,藥物的代謝速率是影響血藥濃度的最主要因素,而藥物的代謝速率和代謝方式則是由特定的藥物代謝酶所決定的。 細(xì)胞色素氧化酶P450是人類肝臟中一種重要藥物代謝酶系,能代謝多種內(nèi)源性底物、藥物和外源性化合物,是目前研究最多的藥物代謝反應(yīng)的第I相酶,其代謝的藥物約占肝臟代謝藥物的40%。CYP2D6是細(xì)胞色素氧化酶P450酶系中的一個(gè)重要成員,是最先被發(fā)現(xiàn)具有基因多態(tài)性的藥物代謝酶,也是迄今為止發(fā)現(xiàn)最具有遺傳多態(tài)性特征的代謝酶。已發(fā)現(xiàn)CYP2D6有70多種突變基因,參與代謝了臨床上20%-25%的處方藥物,而且CYP2D6基因多態(tài)性具有很大的種族差異,其多態(tài)性在不同種族之間呈多元化的分布。 經(jīng)科學(xué)研究證明,相同劑量的藥物在不同種族、不同民族,甚至是不同個(gè)體之間的代謝是不同的,這種代謝差異性是藥物毒性及不良反應(yīng)的主要因素,而藥物代謝酶基因遺傳多態(tài)性是這些不良的醫(yī)療效應(yīng)產(chǎn)生的主要內(nèi)在因素之一。 本研究在建立中國(guó)少數(shù)民族基因組DNA樣本庫(kù),保護(hù)現(xiàn)有少數(shù)民族遺傳資源基礎(chǔ)上,利用維吾爾族基因組DNA樣本庫(kù),通過(guò)現(xiàn)代分子生物學(xué)技術(shù),對(duì)藥物代謝酶基因CYP2D6在維吾爾族人群中的多態(tài)性分布規(guī)律進(jìn)行檢測(cè),從而為了解各民族藥物代謝水平的差異,優(yōu)化治療劑量,減少藥物不良反應(yīng)奠定充分實(shí)驗(yàn)基礎(chǔ)。 [研究方法] 在中央民族大學(xué)新入學(xué)學(xué)生中,通過(guò)問(wèn)卷調(diào)查,篩選三代以內(nèi)無(wú)外族通婚史的少數(shù)民族健康成年人,在知情同意的原則下,按照臨床采血標(biāo)準(zhǔn)進(jìn)行采血,通過(guò)非離心柱法提取全血基因組DNA。按照人類遺傳資源標(biāo)準(zhǔn)編碼統(tǒng)一編號(hào),并通過(guò)瓊脂糖電泳及紫外分光光度計(jì)測(cè)定,剔除斷裂及降解的DNA,剔除純度較低樣本,超低溫保存,建立了中國(guó)少數(shù)民族基因組DNA樣本庫(kù)。 目前對(duì)CYP2D61-5號(hào)外顯子實(shí)驗(yàn)已經(jīng)就緒,本實(shí)驗(yàn)分析CYP2D66-9號(hào)外顯子突變情況及多態(tài)性分布規(guī)律。以維吾爾族人群全基因組DNA為模板擴(kuò)增CYP2D66-9號(hào)外顯子,采用直接測(cè)序法對(duì)各樣本進(jìn)行了序列測(cè)定,利用生物信息學(xué)軟件對(duì)突變位點(diǎn)進(jìn)行篩查,并對(duì)突變位點(diǎn)的多態(tài)性分布規(guī)律進(jìn)行小規(guī)模掃描。在此基礎(chǔ)之上,通過(guò)采用點(diǎn)突變PCR實(shí)驗(yàn)方法以CYP2D6CDS為模板獲得了2444-2445insG、2456GA、4124GC的三個(gè)突變位點(diǎn),并采用雙酶切、鏈接和轉(zhuǎn)化實(shí)驗(yàn)構(gòu)建了三個(gè)突變位點(diǎn)的真核表達(dá)質(zhì)粒。 [研究結(jié)果] 1.中國(guó)少數(shù)民族DNA樣本庫(kù)的建立 共采集血液樣本1800例,其中包括漢族501例、維吾爾族145例。初步建成包括朝鮮族,蒙古族,傣族,哈薩克族,維族,苗族,回族,藏族,土家族,彝族,侗族,羌族,壯族,瑤族,德昂族,納西族,錫伯族,白族,柯爾克孜,滿族,哈尼族,布依族,仡佬族,畬族,黎族,水族,阿昌族,漢族,烏孜別克族,裕固族等37個(gè)民族的1494個(gè)基因組DNA樣本。 2.CYP2D6基因在維吾爾人群中的突變位點(diǎn)篩查 我們?cè)谥袊?guó)維吾爾族人群中對(duì)CYP2D6外顯子6-9篩查中得到2個(gè)已報(bào)導(dǎo)的等位基因型和5個(gè)未報(bào)道的等位基因型。已報(bào)導(dǎo)的等位基因分別是CYP2D6*2和CYP2D6*10D,它們的分布頻率分別為10%和15%。5個(gè)未報(bào)道的新的等位基因型,其分布頻率介于5%到10%之間。 3.CYP2D6突變位點(diǎn)的構(gòu)建 采用點(diǎn)突變方法,利用PCR技術(shù),以pEGFP-N1為載體,成功構(gòu)建了CYP2D6CDS和2444-2445insG、2459GA、4124GC三個(gè)突變位點(diǎn)的真核表達(dá)體系。 [結(jié)論] 1.中國(guó)少數(shù)民族基因組DNA樣本庫(kù)初步成功建立。 2.CYP2D6基因在中國(guó)維吾爾族人群分布頻率與該基因已報(bào)道的其他民族中分布特點(diǎn)存在顯著差異。 3.構(gòu)建了CYP2D6CDS和2444-2445INSG、2459GA、4124GC三個(gè)突變位點(diǎn)的真核表達(dá)體系。
[Abstract]:[purpose and significance]
The drug is usually combined with specific targets and produce the effects of drugs, so there is a close relationship between drug concentration and drug effect target, and target parts of the drug concentration and blood concentration are in a dynamic balance. The blood concentration of not only depends on the dosage of the drug, but also closely related with the pharmacokinetic parameters. Understanding the pharmacokinetic parameters of individual or group, will be able to predict target parts of the drug concentration, the individual administration possible. In a variety of pharmacokinetic parameters, drug metabolism rate is the main factor affecting the blood concentration and the metabolic rate and metabolism of drug is determined by the specific drug metabolizing enzymes.
Cytochrome P450 is an important drug metabolizing enzymes in human liver, metabolism of many endogenous and exogenous substrates, drug compounds, and is currently on most drug metabolic reactions in the phase I enzyme, drug metabolism about liver metabolism of the drug 40%.CYP2D6 is an important member of the cytochrome P450 enzyme system the is the first to be found with drug metabolizing enzyme gene polymorphism, so far found the genetic polymorphism of metabolic enzyme has the most characteristic. CYP2D6 has been found 70 kinds of mutant genes involved in the metabolism of the 20% clinical -25% prescription drugs, and CYP2D6 gene polymorphism of great ethnic differences, its polymorphism a diversified distribution between different races.
The research shows that the drug in the same dose of different races, different ethnic groups, even between different individuals metabolism is different, the metabolic difference is a major factor in drug toxicity and adverse reactions, and drug metabolizing enzymes genetic polymorphism is one of the main factors causing these adverse health effects.
This study was to establish a minority genomic DNA samples Chinese, protection of the existing ethnic genetic resources, utilization of Uygur genomic DNA samples, through modern molecular biology techniques to detect the polymorphism of drug metabolizing enzyme gene CYP2D6 in Uygur population of the distribution, so as to understand the differences in the national drug metabolism level. To optimize the therapeutic dose, reduce adverse drug reactions to lay adequate experimental basis.
[research methods]
Minzu University of China in the newly enrolled students, through questionnaire survey, screening within three generations of minority health adults without exogamy history, under the principle of informed consent, collected in accordance with the standard clinical blood, through a centrifugal column method for genomic DNA. extraction of human genetic resources in accordance with the standard uniform number encoding, and by agarose gel electrophoresis and ultraviolet spectrophotometer, excluding breaks and degradation of DNA, excluding the low purity samples, cryopreservation, established the genomic DNA samples of minority Chinese.
The exon CYP2D61-5 experiment have been completed, the experimental analysis of CYP2D66-9 exon mutation and polymorphism distribution in the Uygur population. The whole genome DNA as template to amplify the CYP2D66-9 exon of each sample were sequenced by direct sequencing and software for mutation screening by using biological information, and small scale scan polymorphism distribution of mutations. On this basis, by using point mutation PCR experimental method using CYP2D6CDS as template to obtain 2444-2445insG, 2456GA, 4124GC three process variable sites, and the use of double enzyme, links and transformation experiments built three mutation sites of eukaryotic expression plasmid.
[results]
The establishment of the DNA sample library of 1. ethnic minorities in China
Blood samples were collected in 1800 cases, including 501 cases of Han and Uygur 145 cases. Initially built including Korean, Mongolian, Uygur, Kazak, Dai, Miao, Tujia, Hui, Tibetan, Yi, Qiang, Dong, Zhuang, Yao, Deang, Naxi, Bai, Kirgiz, Xibe. Manchu, Hani, Buyi, Gelao, Yu, Li, aquarium, Achang, Han, Uzbek Buick, 1494 genomic DNA samples of Yugur and other 37 ethnic groups.
Mutation site screening of 2.CYP2D6 gene in Uygur population
We are on the CYP2D6 exon in China Uygur population 2 reported allele and 5 unreported allele 6-9 screening. The reported alleles were CYP2D6*2 and CYP2D6*10D, the frequency distribution of them were 10% and 15%.5 were not reported in the new allele the distribution of genotype frequencies between 5% to 10%.
Construction of 3.CYP2D6 mutation site
By using the point mutation method and using PCR technology and pEGFP-N1 as carrier, we successfully constructed the eukaryotic expression system of CYP2D6CDS and 2444-2445insG, 2459GA and 4124GC three mutation sites.
[Conclusion]
1. the DNA sample library of Chinese minority genomes has been successfully established.
The distribution frequency of the 2.CYP2D6 gene in the Uygur population in China is significantly different from that of the other ethnic groups reported by the gene.
3. the eukaryotic expression system of three mutation sites of CYP2D6CDS and 2444-2445INSG, 2459GA, 4124GC was constructed.

【學(xué)位授予單位】：中央民族大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R394

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