【作者】 李晋新；

【导师】 程祖亨；

【作者基本信息】 新疆医科大学 ， 心血管内科， 2009， 博士

【摘要】 背景:炎症是组织应对创伤或感染所引发的一系列反应,是细胞和分子之间复杂的相互作用,最终使机体恢复生理平衡和促进组织修复,个体对感染和创伤的不同反应是由基因决定的。最近的研究中认为衰老伴随着慢性低水平的炎症反应状态,血清炎症介质增加到正常值的2-4倍可以清楚地表明这一点。这种慢性持续性低水平的炎症反应状态有多种因素的参与,其中慢性抗原负荷似乎发挥了最重要的作用,通过激活巨噬细胞和淋巴细胞而影响免疫系统,这种“前炎症反应状态”与遗传背景相互作用,有可能触发多种年龄相关性炎症性疾病,如动脉粥样硬化等的发病。因此,分析免疫反应中关键因子的基因的多态性可能明确年龄相关性炎症性疾病如动脉粥样硬化的病理生理机制。另一方面,百岁老人的特点是明显的推迟或免于罹患年龄相关性疾病,百岁老人后代存活至100年的可能性较高,而易于罹患年龄相关性疾病,如心血管疾病(CVD)和具有心血管病危险因素者长期存活的可能性较低,而且与心血管疾病密切关联的基因可能在长寿中发挥相反的作用。因此,对百岁老人的研究可以用来揭示与炎症反应密切相关的基因在成功和不成功的衰老中发挥的作用。百岁老人代表着寿命的极端情况,是进行人类长寿研究最佳的人群。新疆和田地区维吾尔族百岁老人数量和相对比例均高于全国平均水平,是世界四大长寿区之一。并且由于环境、生活特点及风俗习惯,形成了特殊的遗传隔离群,因此被称之为自然长寿人群,是长寿遗传学研究的难得资源。长寿的遗传学研究方法,除常用的比较长寿者和对照人群之间单个多态性基因型分布的研究方法外,连锁不平衡和单倍型分析逐渐成为研究寿命这一受多基因及基因环境相互作用共同控制的复杂表型更为有效的遗传研究方法,对关联分析尤其复杂疾病的关联研究极为有用。目的:探索HSP70基因的三种常见的遗传变异包括A1267G(rs1061581,外显子1),G190C(rs1043618,5′端非编码区)和T2437C(rs2227956,外显子2)的多态性对自然长寿的影响。并探讨TNFβ基因外显子+252G/A和TNFα基因启动子区-863C/A以及TNFα基因启动子区-308G/A多态性并分析其单倍型与新疆和田地区维吾尔族自然长寿之间的关系。方法:以新疆和田地区191名年龄90岁以上的健康维吾尔族自然长寿个体为研究对象,以53名地域、民族、性别相匹配的无长寿家族史,在75岁前自然死亡的个体为对照,进行流行病学调查。测定身高、体重、收缩压(systolic blood pressure,SBP)和舒张压(diastolic blood pressure,DBP)、总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)、载脂蛋白A(apolipoprotein A,ApoA)和血糖(blood glucose,BG)水平。提取白细胞基因组DNA,分别采用PCR(polymerase chain reaction)、PCR-限制片段长度多态性(PCR-restriction fragment length polymorphism,PCR-RFLP)和PCR直接测序等技术对HSP70基因A1267G、G190C、T2437C多态性和TNFβ基因+252G/A以及TNFα基因启动子区-308G/A、-863C/A多态性进行基因分型,并进行单倍型分析。结果:我们研究了热休克蛋白70(HSP70)家族的三个等位基因(A1267G,G190C和T2437C)与新疆和田地区维吾尔族自然长寿的关联。这是一项以191例90岁以上健康的长寿老人和53例同地区的自然死亡年龄在65-70岁个体作对照组的病例对照研究。分析采用EH/EH+程序,用MDR软件分析基因和基因的相互作用。两组中所有的研究变量符合Hardy-Weinberg平衡。所有等位基因的基因型和等位基因频率在长寿组和对照组间的分布无显着差异,与此相反,单倍型分析表明,单倍型A-C-G(按A1267G,T2437C和G190C顺序)和A-T-C在长寿组的分布频率较对照组增高,差异具有显著性(P=0.026和0.017),并分别表现出3.46和4.51倍使寿命增加的趋势。单倍型的结果由交互作用分析进一步加强,G190C和T2437C对促进长寿显示出更为优化的相互作用。综上,我们的研究结果表明,热休克蛋白70家族常见的基因变异在维吾尔族长寿中发挥作用。TNFβ基因外显子+252G/A,TNFα基因启动子-308G/A和TNFα启动子区-863C/A多态性各等位基因、基因型及单倍型与新疆和田维吾尔族自然长寿无关。基因多态性分布不仅存在种族差异,在同一民族之间还存在地域差异。在新疆和田维吾尔族中,存在长期遗传隔离所造成的TNFβ基因外显子+252G/A,TNFα基因启动子-308G/A和TNFα启动子区-863C/A多态性间的特有的分布特点。将3个多态性同时纳入单倍型分析时,在长寿组和对照组均有8种单倍型,分别是A-G-C、G-G-C、A-G-A、G-A-C、A-A-C、G-G-A、G-A-A、A-A-A其中5种为常见单倍型,两组中均以A-G-C为主要单倍型。但均未发现与长寿相关,且交互作用研究并未发现各位点间存在着相互作用。结论:1.HSP70基因多态性可能与长寿相关。热休克蛋白(HSP)三个多态性位点(+1267G/A,+190 G/C和+2437 T/C)的基因型和等位基因频率在长寿组和正常对照组间的差异。单倍型A-C-G和A-T-C(按A1267G,T2437C和G190C的顺序)具有使寿命更长的遗传倾向,而单倍型A-C-C具有截然相反的趋势。交互效应的分析结果表明G190C和T2437C位点间可能有很强的协同效应,从单倍体和其相互作用的结果看来,我们指出这两个基因型的遗传结构中具有一个突变等位基因(190G-2437C和190C-2437T)者更趋向长寿,而另外的突变组合(190C-2437C)不利于长寿。这可能表明这两个突变位点间存在着相互作用。2.对TNF基因多态性的研究显示,+252G/A,-308G/A和-863C/A 3个多态性位点基因型及等位基因的分布在长寿与对照组之间均无统计学差异。3个多态性不同基因型和单倍型在长寿组与对照组中的分布均无统计学差异,未发现具有交互作用的基因位点。更多还原

【Abstract】 Backgroud.Inflammation is considered a response set by the tissues in response to injury elicited by trauma or infection.It is a complex network of molecular and cellular interactions that facilitates a return to physiological homeostasis and tissue repair.The individual response against infection and trauma is also determined by gene variability. Ageing is accompanied by chronic low-grade inflammation state clearly showed by 2-4-fold increase in serum levels of inflammatory mediators.A wide range of factors has been claimed to contribute to this state;however,the most important role seems to be played by the chronic antigenic stress,which affects immune system thorough out life with a progressive activation of macrophages and related cells.This pro-inflammatory status,interacting with the genetic background,potentially triggers the onset of age-related inflammatory diseases as atherosclerosis.Thus,the analysis of polymorphisms of the genes that are key nodes of the natural immunity response might clarify the patho-physiology of age-related inflammatory diseases as atherosclerosis.On the other hand,centenarians are characterized by marked delay or escape from age-associated diseases that,on average,cause mortality at earlier ages.In addition,centenarian offspring have increased likelihood of surviving to 100 years and show a reduced prevalence of age-associated diseases,as cardiovascular disease(CVD) and less prevalence of cardiovascular risk factors.So,genes involved in CVD may play an opposite role in human longevity.Thus,the model of centenarians can be used to understand the role of these genes in successful and unsuccessful ageing.The centenarian,who represents the extremity of longevity,was the optimal population of researching the human longevity.The quantity and relative ratio of centenarian in Xinjiang Hetian were over the average level of whole country,so Hetian was classified as one of four longevity regions in the world.Furthermore,because of environment,living characteristics,customs and habits,the Uygur who lived in this region became genetic isolation population and belonged to the natural longevity.Thereby,they were the value resource of gendetic study about longevity.Besides the common methods which compared the genotype distribution of single polymorphism between the longevities and the controls,the genetic methods of longevity study include the linkage disequilibrium and haplotype analysis,which became increasingly the effective methods of studying the life-span that was the complex phenotype controlled by multiple genes and gene-environment interaction.The linkage disequilibrium and haplotype analysis were valuable to association analysis,especially the association study of complex diseases.Objectives:To investigate the association between polymorphisms(A1267G, G190C andT2437C) and their haplotype within heat shock protein(HSP) gene,as well as polymorphisms(+252G/A,-863C/A and-308G/A) and their haplotype within the tumor necrosis factor(TNF) gene,and natural longevity in Xinjiang Uygur population.Methods:191 healthy Uygur individuals over 90 years in Xinjiang Hetian were recruited.And 53 area-,nationality-,gender-matched individuals who had no longevity family history and died in their 75 years were studied as control subjects.Their height, weight,systolic blood pressure(SBP) and diastolic blood pressure(DBP),serum total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),apolipoprotein A(ApoA) and blood glucose(BG) were measured.Genome DNA was extracted from white blood cells.Using polymerase chain reaction(PCR),PCR-restriction fragment length polymorphism(PCR-RFLP) and direct sequencing technique,we tested the polymorphisms of the polymorphisms of A1267G、G190C and T2437C within H SP70 gene,the polymorphisms of+252G/A within TNFβand the polymorphisms of -308G/A and-863C/A within TNFαgene,moreover, performed the linkage disequilibrium and haplotype analysis.Results:We investigate the association of three variants(A1267G,G190C and T2437C) in heat shock protein 70(Hsp70) family with natural longevity in a Xinjiang Hetian Uygur population.A case-control study was conducted in 191 healthy individuals aged above 90 years,and 53 naturally-died persons aged 65-70 years.Promoter activity was evaluated by luciferase reporter assays.Data were analyzed using EH/EH+ program for hypotype prediction and MDR software for gene-gene interaction.All studied variants satisfied Hardy-Weinberg equilibrium in each group.In single-locus analysis,no significant differences were found between longevities and controls in the genotype/allele distributions of all variants.In contrast,haplotype analysis indicated that haplotypes A-C-G(in order of A1267G,T2437C and G190C) and A-T-C were more prevalent in longevities than controls(P=0.026 and 0.017),and conferred a 3.46- and 4.51-fold increased tendency for longevity,respectively.Haplotype results were further strengthened by interaction analysis,suggesting an optimal model with G190C and T2437C exerting an interacting effect on longevity.Taken together,our findings suggested that common genetic variants in Hsp70 family might contribute interactively to longevity in Uygur.The variants(+252G/A,-308G/A and -863C/A) in TNF gene,including alleles, genotype and haplotype show no association with longevity.The gene polymorphism exist not only in ethinity but also in area.Special charactoristics exist in polymorphism of +252G/A,-308G/A and -863C/A with Hetian Uygur.There are 8 haplotypes of the TNF gene,that is,A-G-C、G-G-C、A-G-A、G-A-C、A-A-C、G-G-A、G-A-A and A-A-A.The most common is A-G-C.There is no association with longevity.Conclusions:AA genotype of promoter-2437G/A polymorphism within HSP gene was the advantageous factor of longevity.A-C-G and A-C-T haplotype of A1267G,T2437C和G190C polymorphisms was the beneficial factor of longevity.. Furthermore,there was the extensive linkage among G190C and T2437C,resulted from long-term genetic isolation in Uygur in Xinjiang Hetian.There was no correlation between the alleics,genotypes and haplotypes of polymorphisms within TNF gene,Furthermore,there was no linkage between+252G/A, -308G/A and -863C/A,resulted from long-term genetic isolation in Uygur in Xinjiang Hetian.And the result of-863C/A polymorphism suggested the base sequences of this polymorphism had race specificity更多还原