【作者】 龚志成；

【导师】 刘昭前；

【作者基本信息】 中南大学 ， 药理学， 2012， 博士

【摘要】 遗传药理学(pharmacogenetics)是研究人体遗传变异引起的药物代谢酶、药物转运体和药物作用靶点功能异常,导致药物代谢和效应个体差异的一门科学。遗传药理学从基因角度来解释药物治疗效应个体差异和不良反应的根本机制,运用分子生物学的最新技术和方法来研究药物的作用,把从分子水平的学科到临床水平的学科,包括药理学、生理学、遗传学、基因组学、临床医学、流行病学、统计学、生物信息学、生物计算机学等多学科联合到一起来阐明药物的作用和作用机理。药物反应个体差异是临床药物治疗中常见的普遍现象。引起药物反应个体差异的原因很多,有性别、年龄、体重、伴随的疾病等,遗传因素是十分重要的一个因素。遗传药理学研究发现,药物代谢酶、受体和转运体活性的改变是导致临床上药物治疗中出现药物反应个体差异常见原因,药物代谢酶、受体和转运体基因的遗传多态性是药物作用个体差异的重要决定因素。2型糖尿病也称非胰岛素依赖型糖尿病,占糖尿病患者的90%左右,其发病率逐年增加。目前全球2型糖尿病人已超过1.7亿,世界卫生组织预计2025年将会突破3亿,而发病年龄呈现年轻化趋势。2型糖尿病的发病机制包括：遗传易感性,胰岛B细胞功能障碍以及胰岛素抵抗现象。其治疗的短期目标是控制血糖,长期目标是预防相关并发症的发生与发展。其基础治疗方案主要由运动和饮食构成,但是药物治疗和血糖监测往往也非常关键。瑞格列奈适用于2型糖尿病病人,具有起效快、作用时间短等特点。瑞格列奈作用的机理尚不完全清楚,可能与药物的受体、药物代谢酶、药物转运体及糖尿病相关基因的多态性对药物作用的影响有关。神经分化因子1(neurogenic differentiation1, NeuroD1),又叫p细胞E盒反式激活因子2(β-cellE-box transactivator2),作为正向调控蛋白NeuroD1/BETA2对胰腺p细胞的分化及正常功能具有举足轻重的地位。早期发现NeuroD1/BETA2基因45位密码子发生突变,导致氨基酸Ala被Thr替代,该突变发生在211位核苷酸处(G>A),这一变异位于该基因的功能域,影响基因的转录活性和胰岛p细胞的分化和重建能力。对于该多态性与中国2型糖尿病的相关性不明。转录因子配对盒4(the paired box gene4, Pax4)主要功能为胰腺发育的转录抑制因子,在胰腺p细胞的分化和发育过程中发挥重要作用。PAX4基因敲除小鼠胰腺成熟的胰岛素分泌细胞和生长抑素分泌细胞完全缺失,而分泌胰高糖素的α细胞增生。PAX4(the paired box gene4)基因定位于人染色体7q32,包括9个外显子和8个内含子。日本人的研究结果提示R121W(Arg121Trp)与2型糖尿病相关,且对胰岛p细胞的功能有影响。另一报道显示,携带突变纯合子121W的2型糖尿病患者缺乏胰岛素分泌第一时相。该基因多态与中国2型糖尿病的相关性未见报道。全基因组关联分析研究(Genome-wide association studies, GWASs)和meta分析发现多个新的T2DM的易感位点,这些区域可能在胰岛素的分泌和胰腺B细胞功能中发挥重要作用。其中KQT亚科-钾离子电压门控通道1(potassium voltage-gated channel, KQT-like subfamily, member1, KCNQ1)基因在多个人群的研究均发现,rs2237892和rs2237895多态与2型糖尿病相关性强。KvLQT1蛋白属于电压门控钾通道蛋白家族,KCNQ1基因广泛表达于心脏、胰腺、前列腺、肾脏、小肠和外周血白细胞中。该基因突变可能损伤胰岛p细胞的功能,从而影响胰腺分泌。瑞格列奈是一种胰岛素促分泌剂,又名“膳食葡萄糖调节剂”,被临床广泛的用于治疗经饮食控制、控制体重及锻炼后仍不能有效控制血糖高水平的2型糖尿病患者。瑞格列奈与磺酰脲类受体结合,关闭胰腺β细胞上ATP敏感性钾通道,开放钙通道,钙内流增加,促进胰岛素的分泌。瑞格列奈改善早相胰岛素分泌,起效快,作用时间短,主要用于控制餐后高血糖水平。临床观察发现瑞格列奈出现显著的个体疗效差异,常导致药物疗效不佳或失效,其具体机制不明。本实验旨在研究中国汉族人群NeuroD1/BETA2基因Ala45Thr、 PAX4基因R121W和KCNQ1基因rs2237892和rs2237895多态性的中国人群等位基因、基因型频率分布特征及其与代谢相关指标的关系,了解该基因多态性与2型糖尿病的易感相关性及对瑞格列奈疗效的影响。本课题的主要研究结果如下：NeuroD1/BETA2基因Ala45Thr位点与T2DM易感性显著相关,并且能影响瑞格列奈治疗T2DM的疗效。PAX4基因R121W位点可能不是T2DM的独立危险因子,但是能影响瑞格列奈的疗效。KCNQ1基因rs2237892位点、rs2237895位点与T2DM相关,且均对瑞格列奈的疗效有影响。1. NeuroD1/BETA2基因Ala45Thr位点与2型糖尿病易感性显著相关,带突变基因型的个体更易患2型糖尿病。2. NeuroD1/BETA2基因Ala45Thr位点野生基因型A/A组的FINS水平和PINS水平显著高于突变基因型(A/T+T/T)组。3.经瑞格列奈治疗后每天3mg口服8周治疗后,携带NeuroD1/BETA2基因Ala45Thr位点A/A基因型的2型糖尿病患者FPG下降水平和PPG下降水平显著高于A/T+T/T基因型患者,NeuroD1/BETA2基因Ala45Thr位点影响瑞格列奈治疗2型糖尿病的疗效。4. PAX4基因R121W位点等位基因和基因型频率在2型糖尿病组和健康对照组间的分布无显著性差异。5. PAX4野生基因型R/R组的PINS水平显著高于突变基因型(R/W+W/W)组。PAX4基因R121W位点多态性与2型糖尿病患者的餐后胰岛素水平相关。6.经瑞格列奈治疗后每天3mg口服8周治疗后,携带PAX4基因R121W位点R/R基因型的2型糖尿病患者PPG下降水平显著高于R/W+W/W基因型患者。PAX4基因R121W位点多态性影响瑞格列奈的疗效。7.2型糖尿病组的KCNQ1基因rs2237892位点TT基因型频率和T等位基因频率显著低于健康对照组。2型糖尿病组的KCNQ1基因rs2237895位点C等位基因频率显著高于健康对照组。8. KCNQ1基因rs2237892位点CC, CT和TT基因型组的FINS. HOMA-IR值三组之间有显著差异,且表现为基因剂量效应。KCNQ1基因rs2237895位点AA,AC和CC基因型组的FPG. PPG和HOMA-IR值三组之间有显著差异,且表现为基因剂量效应。9. KCNQ1基因rs2237892位点不同基因型组患者经瑞格列奈治疗后的PPG值三组之间有显著性差异,对瑞格列奈的疗效有影响。KCNQ1基因rs2237895位点不同基因型组患者经瑞格列奈治疗后的FINS值三组之间有显著性差异,对瑞格列奈的疗效有影响。本课题为2型糖尿病的病因研究及合理用药的机理进行初步探索。为2型糖尿病临床早期预测、诊断和治疗提供新的契机,并为糖尿病的个体化药物治疗提供实验依据。更多还原

【Abstract】 Pharmacogenetics refers to drug metabolism, effective group and individual difference abnormality which mainly caused by genetic variance and disfunction of drug-metabolizing enzyme, drug transport or target proteins. Pharmacogenetics aims to explain the basic mechanisms of drug therapeutic efficacy individual difference and adverse reaction from point of view of genetic polymorphisms. From molecular level to clinical level subjects, it combines pharmacology, physiology, hereditism, genomics, clinical medicine, epidemiology, statistics, bioinformatics and biocomputer to elucidate medicine functions and its mechanisms. Individual difference of drug reaction is a common phenomenon in clinical drug use. The reasons include gender, age, weight and accompanied diseases and so on, among them, genetic factors is very important. Pharmacogenetics studies indicate that activity change of drug-metabolizing enzymes, receptors and transporter is the major mechanism for individual differences in drug response, and inherited variation in activities of drug-metabolizing enzymes, receptors and transporter results in inter-individual differences in drug metabolism, disposition and efficacy.Type2diabetes mellitus (T2DM), also called noninsulin-dependent diabetes mellitus, is a complex metabolic disease. Type2diabetes mellitus accounts90%of all diabetes and rapidly increasing prevalence year by year. Now more than170million subjects worldwide are type2diabetes mellitus patients. The world healthy organization estimates that the number of type2diabetes mellitus patients will be more than3billions in2025and age of onset shows make more youthful tendency. The pathogenesy of type2diabetes includes hereditary susceptibility, fi-cell disfunction in pancreas islet and insulin resistance. NeuroD1 (neurogenic differentiation1), also called β-cell E-box transactivator2, it very important for pancreas β-cell differentiation and normal function as positive regulation protein. It has been reported that NeuroDl/BETA2gene had a polymorphism in211nucleotide position (G>A), which caused Ala substituted by Thr. This polymorphism locates in function domain and may influence the gene transcription activity and the differentiation and rebuild ability of pancreas B-cell. The association between NeuroD1/BETA2gene Ala45Thr and type2diabetes mellitus in Chinese is not clear until now.The paired box gene4plays very important role in pancreas B-cell differentiation and development which as a transcript inhibition factor. PAX4gene knock-out mouse showed insulin secretion cells and growth hormone release inhibiting hormone secretion cells absolutely absence in full-blown pancreas, but glicentin secreted a-cell were proliferation. PAX4gene located in chromatosome7q32combined9exons and8introns. It has been reported PAX4gene R121W polymorphism associated with type2diabetes in Japanese and effected on pancreas B-cell function. Another study results showed patients with mutate homozygote121W was short of the first phase of insulin secretion. We don’t know the association of PAX4gene R121W polymorphism and Chinese type2diabetes mellitus.Genome-wide association studies (GWASs) and meta-analyses identified several new susceptibility gene polymorphisms of type2diabetes mellitus. These gene polymorphisms play important roles in insulin secretion and pancreas B-cell function. KCNQ1(potassium voltage-gated channel, KQT-like subfamily, member1) gene rs2237892and rs2237895polymorphisms associated with type2diabetes mellitus in several populations. It belongs to potassium voltage-gated channel family and expressing in heart, pancreatic gland, prostate, kidney, small intestine and peripheral blood leucocyte. The KCNQ1gene polymorphisms affect the function and insulin secretion of β-cell.Repaglinide is an insulin secretagogue agent, also called Dietary glucose regulator. It is widely used in type2diabetes patients who have higher blood glucose after alimentary control, body weight control and exercise. Repaglinide binds with sulfonylurea receptor, then blocks potassium voltage-gated channel and open calcium voltage-gated channel, causing increasing insulin secretion. Repaglinide is mainly used in control postprandial hyperglycemia and improve the first phase insulin secretion. Repaglinide therapeutic efficacy shows individual differences.This study investigated whether NeuroDl/BETA2gene Ala45Thr polymorphism, PAX4gene R121W polymorphism and KCNQ1gene rs2237892and rs2237895polymorphisms are associated with type2diabetes and repaglinide efficacy in Chinese T2DM patients.The present series of studies have found that:1. NeuroD1/BETA2gene Ala45Thr polymorphism was associated with the development of type2diabetes mellitus.2. Patients with mutated NeuroDl/BETA2gene Ala45Thr polymorphism showed higher FINS and PINS levels than that in A/T+T/T individuals.3. NeuroD1/BETA2gene Ala45Thr polymorphism affects repaglinide therapeutic efficacy. Patients with A/A genotypic NeuroDl/BETA2gene showed higher FPG DV and PPG DV values than that in A/T+T/T individuals.4. The genotypic and allelic frequencies of PAX4gene R121W polymorphism shows no differences between type2diabetes group and healthy controls.5. Patients with PAX4gene R/R polymorphism showed higher PINS levels than that in R/W+W/W individuals. 6. PAX4gene R121W polymorphism affects repaglinide therapeutic efficacy. T2DM Patients with R/R genotypic PAX4gene showed higher PPG DV values than that in R/W+W/W individuals.7. KCNQ1gene rs2237892and rs2237895polymorphisms were associated with type2diabetes and showed gene dosage effect.8. KCNQ1gene rs2237892polymorphism was associated with the values of FINS and HOMA-IR and KCNQ1gene rs2237895polymorphism affected markedly the values of PPG and HOMA-IR in T2DM patients.9. KCNQ1gene rs2237892and rs2237895polymorphisms affected repaglinide therapeutic efficacy and showed gene dosage effect in T2DM patients.The present study has provided novels susceptibility gene locus for type2diabetes mellitus. We try to explain the possible mechanism of individual differences in repaglinide therapeutic efficacy.更多还原