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

【摘要】：[目的與意義] 藥物通常是和特定的靶點結(jié)合而產(chǎn)生藥物效應,因此藥物效應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力學參數(shù)密切相關(guān)。因此如能了解個體或群體的藥物代謝動力學參數(shù),就能有效預測靶部位的藥物濃度,使個體化給藥成為可能。在各種藥物動力學參數(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的第I相酶,其代謝的藥物約占肝臟代謝藥物的40%。CYP2D6是細胞色素氧化酶P450酶系中的一個重要成員,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)療效應產(chǎn)生的主要內(nèi)在因素之一。 本研究在建立中國少數(shù)民族基因組DNA樣本庫,保護現(xiàn)有少數(shù)民族遺傳資源基礎上,利用維吾爾族基因組DNA樣本庫,通過現(xiàn)代分子生物學技術(shù),對藥物代謝酶基因CYP2D6在維吾爾族人群中的多態(tài)性分布規(guī)律進行檢測,從而為了解各民族藥物代謝水平的差異,優(yōu)化治療劑量,減少藥物不良反應奠定充分實驗基礎。 [研究方法] 在中央民族大學新入學學生中,通過問卷調(diào)查,篩選三代以內(nèi)無外族通婚史的少數(shù)民族健康成年人,在知情同意的原則下,按照臨床采血標準進行采血,通過非離心柱法提取全血基因組DNA。按照人類遺傳資源標準編碼統(tǒng)一編號,并通過瓊脂糖電泳及紫外分光光度計測定,剔除斷裂及降解的DNA,剔除純度較低樣本,超低溫保存,建立了中國少數(shù)民族基因組DNA樣本庫。 目前對CYP2D61-5號外顯子實驗已經(jīng)就緒,本實驗分析CYP2D66-9號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為模板擴增CYP2D66-9號外顯子,采用直接測序法對各樣本進行了序列測定,利用生物信息學軟件對突變位點進行篩查,并對突變位點的多態(tài)性分布規(guī)律進行小規(guī)模掃描。在此基礎之上,通過采用點突變PCR實驗方法以CYP2D6CDS為模板獲得了2444-2445insG、2456GA、4124GC的三個突變位點,并采用雙酶切、鏈接和轉(zhuǎn)化實驗構(gòu)建了三個突變位點的真核表達質(zhì)粒。 [研究結(jié)果] 1.中國少數(shù)民族DNA樣本庫的建立 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個民族的1494個基因組DNA樣本。 2.CYP2D6基因在維吾爾人群中的突變位點篩查 我們在中國維吾爾族人群中對CYP2D6外顯子6-9篩查中得到2個已報導的等位基因型和5個未報道的等位基因型。已報導的等位基因分別是CYP2D6*2和CYP2D6*10D,它們的分布頻率分別為10%和15%。5個未報道的新的等位基因型,其分布頻率介于5%到10%之間。 3.CYP2D6突變位點的構(gòu)建 采用點突變方法,利用PCR技術(shù),以pEGFP-N1為載體,成功構(gòu)建了CYP2D6CDS和2444-2445insG、2459GA、4124GC三個突變位點的真核表達體系。 [結(jié)論] 1.中國少數(shù)民族基因組DNA樣本庫初步成功建立。 2.CYP2D6基因在中國維吾爾族人群分布頻率與該基因已報道的其他民族中分布特點存在顯著差異。 3.構(gòu)建了CYP2D6CDS和2444-2445INSG、2459GA、4124GC三個突變位點的真核表達體系。
[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.

【學位授予單位】：中央民族大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R394

【參考文獻】

相關(guān)期刊論文 前10條

1 李國昌,陳勇,陳文,王玉華;中國漢族、維吾爾族、哈薩克族右美沙芬氧化代謝多態(tài)性表型研究[J];中國臨床藥理學雜志;2004年02期

2 李芹;王睿;郭雅;裴斐;;中國人群細胞色素P4502D6基因多態(tài)性對曲馬多藥代動力學的影響[J];中國臨床藥理學雜志;2009年04期

3 李文凱;劉清霞;陳放知;駱亞萍;陳慧娟;夏嘉志;陳漢春;;CYP1A1和CYP2D6基因多態(tài)性對白血病發(fā)生的影響[J];基礎醫(yī)學與臨床;2008年06期

4 李宗吉;王健;王惠成;葛立軍;;寧夏回族人群CYP2D6*10等位基因及基因型分布頻率[J];寧夏醫(yī)科大學學報;2009年06期

5 施安國;CYP2D6基因與藥物代謝[J];中國新藥與臨床雜志;2003年08期

6 諸葛堅,余應年;人細胞色素P450前mRNA的可變剪接研究進展[J];中國病理生理雜志;2005年02期

7 雷霆雯,許慶忠,吳寧,李紅,吳青青;中國苗族CYP2D6*10B基因多態(tài)性研究[J];中國公共衛(wèi)生;2004年04期

8 于亮,黃小琴,史荔,史磊,俞建昆,褚嘉yP;中國四個少數(shù)民族九個Y-STR位點基因頻率和單倍型研究[J];中華醫(yī)學遺傳學雜志;2005年03期

9 謝紅光,周宏灝;遺傳藥理學[J];中華醫(yī)學雜志;1997年01期

10 黃小琴,褚嘉yP,林克勤,陶玉芬,俞建昆,史磊,史荔,于亮,石鐵流;建立不同民族永生細胞株質(zhì)量控制方法的探討[J];中國優(yōu)生與遺傳雜志;2005年09期

，

本文編號：1735886