N-乙酰基转移酶1表型与基因多态性的关系及底物调控研究

发布时间：2018-07-17 02:41

【摘要】：背景:N-乙酰基转移酶1(NAT1)是广泛分布于人体内呈多态性表达的酶类。其基因型和表型在人群中分布情况均存在变化。某些药物(对氨基苯甲酸,5-氨基水杨酸)体内经NAT1同工酶代谢,目前认为可以应用这些底物评估不同个体NAT1乙酰化代谢多态性对临床药物治疗疾病的影响。目的:探讨汉族人群中NAT1基因多态性的分布特点,以及基因型和表型之间的相关分析。同时研究对氨基苯甲酸(PABA)对部分表型NAT1活性及NAT1 mRNA的影响。方法:收集140例汉族健康人的外周血,应用PCR(聚合酶链反应)、RFLP(限制性片段长度多态性分析)及多重PCR技术,进行NAT1等位基因分型研究。同时选择PABA作为特异底物检测外周血白细胞中NAT1的活性,代谢产物测定采用高效液相色谱(HPLC)方法。通过提取部分NAT1表型外周血中的淋巴细胞进行培养,观察不同浓度PABA(2、6、18uM)在不同时间(24h、48h、72h)对NAT1活性及NAT1 mRNA水平的影响,NAT1 mRNA分析采用RT-PCR方法。结果:联合应用PCR-RFLP及多重PCR方法可避免多种限制性内切酶之间的相互干扰,可准确区分不同NAT1基因型。在140例检测标本中,NAT1*3,NAT1*4,NAT1*10和NAT1*11的发生频率分别是8.2%,49.6%,40%和2.2%。NAT1*4的发生率明显低于欧美人群,但高于东南亚和非洲人群;而NAT1*10的发生率高于欧美人群,NAT1*3和NAT1*11的发生率较低,与大多数亚洲人群基本一致。从140例检测标本中选择32份不同NAT1基因型进行活性检测,通过计算NAT1内部清除率(Clint)、酶催化反应最大速率(V_(max))和米氏常数(Km)作为评估依据。结果发现,与野生型NAT1*4/*4相比,NAT1*4/*10、NAT1*10/*10以及NAT1*10/*3酶活性属于快代谢表型(p0.05);NAT1*11/*11和NAT1*4/*11酶活性要明显低于NAT1*4/*10、NAT1*10/*10和NAT1*10/*3(p0.05),而与NAT1*4/*3、
[Abstract]:Background: N- acetyltransferase 1 (NAT1) is a polymorphic enzyme widely distributed in human body. The genotypes and phenotypes varied in the population. Some drugs (p-aminobenzoate 5-aminosalicylic acid) have been metabolized by NAT1 isozyme. It is suggested that these substrates can be used to evaluate the effects of different individuals on the clinical therapeutic effects of NAT1 acetylated metabolic polymorphisms. Objective: to investigate the distribution of NAT1 gene polymorphism and the correlation between genotypes and phenotypes in Han population. The effects of p-aminobenzoic acid (PABA) on NAT1 activity and NAT1 mRNA were also studied. Methods: peripheral blood samples were collected from 140 healthy people of Han nationality. The genotyping of NAT1 allele was studied by polymerase chain reaction (PCR) RFLP (restriction fragment length polymorphism analysis) and multiplex PCR technique. At the same time, PABA was selected as the specific substrate to detect the activity of NAT1 in peripheral blood leukocytes, and the metabolites were determined by high performance liquid chromatography (HPLC). The effects of different concentrations of PABA (2 ~ 6 ~ (18) UM) on the activity of NAT1 and the expression of NAT1 mRNA at different time (24 h ~ 48 h ~ 72 h) were studied by RT-PCR. Results: the combination of PCR-RFLP and multiplex PCR could avoid the interference between restriction endonuclease and distinguish different NAT1 genotypes accurately. In 140 samples, the occurrence frequency of Nat 1O 3, Nat 1O 4 and NAT1T 11 were 8. 2% and 2. 2%, respectively, which were significantly lower than those in European and American population, but higher than those in Southeast Asia and Africa, while the incidence of NAT1 10 was lower than that of European and American population, and the incidence rate of NAT1 / Nat 11 was lower than that of European and American population, and the incidence rate of NAT1 / 10 was significantly lower than that of European and American population, but higher than that of Southeast Asia and Africa, respectively, while the incidence of NAT1 / 10 was higher than that of European and American population. Basically consistent with the majority of Asian people. Thirty two different NAT1 genotypes were selected from 140 samples for activity detection. The internal clearance rate (Clint) of NAT1, the maximum rate of enzymatic catalytic reaction (V _ (max) and Michlet constant (km) were calculated as the basis for the evaluation. The results showed that compared with the wild type NAT1 / Nat 4 / 4, the activities of NAT1 / 10 / 10 / 10 and NAT1 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 / 10 of the wild type NAT1 / Nat / 10 / 10 / 10 of Nat 1 / 10 / 10 / 10 / 10 / 10 / 10
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R394

【相似文献】