中国少数民族基因组DNA样本库的建立和CYP2D6在维吾尔族人群中的多态性分布规律

发布时间：2018-04-11 11:38

本文选题：CYP2D6 + PCR　；参考：《中央民族大学》2012年硕士论文

【摘要】：[目的与意义] 药物通常是和特定的靶点结合而产生药物效应,因此药物效应与靶部位的药物浓度有密切的关系,而靶部位的药物浓度又与血药浓度存在着动态平衡。血药浓度不仅取决于药物剂量,而且还与各种药物代谢动力学参数密切相关。因此如能了解个体或群体的药物代谢动力学参数,就能有效预测靶部位的药物浓度,使个体化给药成为可能。在各种药物动力学参数中,药物的代谢速率是影响血药浓度的最主要因素,而药物的代谢速率和代谢方式则是由特定的药物代谢酶所决定的。细胞色素氧化酶P450是人类肝脏中一种重要药物代谢酶系,能代谢多种内源性底物、药物和外源性化合物,是目前研究最多的药物代谢反应的第I相酶,其代谢的药物约占肝脏代谢药物的40%。CYP2D6是细胞色素氧化酶P450酶系中的一个重要成员,是最先被发现具有基因多态性的药物代谢酶,也是迄今为止发现最具有遗传多态性特征的代谢酶。已发现CYP2D6有70多种突变基因,参与代谢了临床上20%-25%的处方药物,而且CYP2D6基因多态性具有很大的种族差异,其多态性在不同种族之间呈多元化的分布。经科学研究证明,相同剂量的药物在不同种族、不同民族,甚至是不同个体之间的代谢是不同的,这种代谢差异性是药物毒性及不良反应的主要因素,而药物代谢酶基因遗传多态性是这些不良的医疗效应产生的主要内在因素之一。本研究在建立中国少数民族基因组DNA样本库,保护现有少数民族遗传资源基础上,利用维吾尔族基因组DNA样本库,通过现代分子生物学技术,对药物代谢酶基因CYP2D6在维吾尔族人群中的多态性分布规律进行检测,从而为了解各民族药物代谢水平的差异,优化治疗剂量,减少药物不良反应奠定充分实验基础。 [研究方法] 在中央民族大学新入学学生中,通过问卷调查,筛选三代以内无外族通婚史的少数民族健康成年人,在知情同意的原则下,按照临床采血标准进行采血,通过非离心柱法提取全血基因组DNA。按照人类遗传资源标准编码统一编号,并通过琼脂糖电泳及紫外分光光度计测定,剔除断裂及降解的DNA,剔除纯度较低样本,超低温保存,建立了中国少数民族基因组DNA样本库。目前对CYP2D61-5号外显子实验已经就绪,本实验分析CYP2D66-9号外显子突变情况及多态性分布规律。以维吾尔族人群全基因组DNA为模板扩增CYP2D66-9号外显子,采用直接测序法对各样本进行了序列测定,利用生物信息学软件对突变位点进行筛查,并对突变位点的多态性分布规律进行小规模扫描。在此基础之上,通过采用点突变PCR实验方法以CYP2D6CDS为模板获得了2444-2445insG、2456GA、4124GC的三个突变位点,并采用双酶切、链接和转化实验构建了三个突变位点的真核表达质粒。 [研究结果] 1.中国少数民族DNA样本库的建立共采集血液样本1800例,其中包括汉族501例、维吾尔族145例。初步建成包括朝鲜族,蒙古族,傣族,哈萨克族,维族,苗族,回族,藏族,土家族,彝族,侗族,羌族,壮族,瑶族,德昂族,纳西族,锡伯族,白族,柯尔克孜,满族,哈尼族,布依族,仡佬族,畲族,黎族,水族,阿昌族,汉族,乌孜别克族,裕固族等37个民族的1494个基因组DNA样本。 2.CYP2D6基因在维吾尔人群中的突变位点筛查我们在中国维吾尔族人群中对CYP2D6外显子6-9筛查中得到2个已报导的等位基因型和5个未报道的等位基因型。已报导的等位基因分别是CYP2D6*2和CYP2D6*10D,它们的分布频率分别为10%和15%。5个未报道的新的等位基因型,其分布频率介于5%到10%之间。 3.CYP2D6突变位点的构建采用点突变方法,利用PCR技术,以pEGFP-N1为载体,成功构建了CYP2D6CDS和2444-2445insG、2459GA、4124GC三个突变位点的真核表达体系。 [结论] 1.中国少数民族基因组DNA样本库初步成功建立。 2.CYP2D6基因在中国维吾尔族人群分布频率与该基因已报道的其他民族中分布特点存在显著差异。 3.构建了CYP2D6CDS和2444-2445INSG、2459GA、4124GC三个突变位点的真核表达体系。
[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.

【学位授予单位】：中央民族大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R394

【参考文献】