【作者】 张伟光；

【导师】 白小涓；

【作者基本信息】 中国医科大学 ， 心血管内科， 2010， 博士

【摘要】 研究背景衰老(ageing, senescence)又称老化,通常是指在正常状况下生物发育成熟后,随年龄增加,自身机能减退,内环境稳定能力与应激能力下降,结构、组分逐步退行性变,趋向死亡,不可逆转的现象。20世纪后期,在老年学研究中引入分子生物学与细胞生物学理论与技术后,研究者们发现,衰老虽然机理复杂,涉及面广,学说虽多,但不外乎遗传与环境两个方面。位于染色体8p-12上的WRN基因突变所致的隐性遗传病Wemer早老综合征(成人型早老症)；同一种属不同个体具有不同衰老表型和不同的寿命；家系调查、双生子以及流行病学研究都表明,衰老是一个高度个体化的过程,衰老与遗传因素密切相关。个体衰老的速度各不相同,随着年龄的增加,人类个体间的差异也在加大。有的人在85岁还有很好的生理和认知功能,但有的人在55岁可能就会出现广泛的认知功能障碍或生理功能失调,时序年龄(Chronological age, CA)不能作为衰老过程准确的指示。从衰老基础和临床研究的目的而言,研究者关心的是个体的衰老变化,目的是筛查衰老高危个体并及时给予干预。而为了解决以上的问题,研究者提出了“生物学年龄(Biological age, BA)"的概念。生物学年龄在个体相对于时序年龄同龄人的功能状态的基础上,评价了个体的功能状态。个体衰老速度不同导致时序年龄和生物学年龄的差异,因此在任何给定的时序年龄,个体间的生物学年龄的值都可能会存在差异,并最终可以预期与个体在衰老过程的结果的时间和／或幅度的差异性相一致。衰老时人体机能下降,而对一些疾病的易感性增强,有人将长寿看做是衰老一个成功的表型,从这个角度而言,某些老年疾病相关基因和长寿相关基因,也可看作是衰老相关基因。很多学者认为衰老和高血压,动脉粥样硬化,痴呆,癌症等疾病一样,并非单一基因决定,而很可能是一个庞杂的衰老相关基因的基因群。研究衰老过程基因的表达谱,筛选新基因,分离、克隆衰老相关基因,弄清其功能、调控、影响因素是弄清衰老机制的一条必由之路。以生物学功能相关基因作为候选基因进行关联研究是当前衰老遗传易感性研究的主要策略。研究表明,基因组序列上的变异,即基因多态性(gene polymorphism)是决定人体对衰老相关疾病易感性与抵抗性、衰老临床表型多样性以及人体对药物治疗反应差异性的重要因素。DNA多态性研究进展可分为3阶段：限制性片段长度多态性、微卫星多态性和单核苷酸多态性(single nucleotide polymorphism, SNP)。SNPs相比于前两代遗传标记其密度高、遗传稳定性好、具有代表性、分布不均匀且其分析易自动化。SNPs的变化,尤其是位于编码区和位于表达调控序列中SNPs的改变,可能引起蛋白质表达的质或量的变化,进而影响蛋白质的结构或表达水平。因而研究SNPs有助于解释个体的表型差异、不同群体和个体对于衰老和长寿的易感性差异。怎样从众多SNPs中,找到确有临床意义的与衰老和衰老疾病相关的SNP位点,是揭示人类衰老致病原因的重要途径之一。胰岛素／胰岛素样生长因子1(insulin/insulin-like growth factor, insulin/IGF-1)途径交互作用于许多种蛋白同时调控大量的基因表达,Sirt1、IL-6、P16及Klotho四个基因或者参与insulin/IGF-1信号途径,或者和insulin/IGF-1信号途径交互作用从而可能影响衰老的进程。IL-6是一个炎性细胞因子,其能影响多个炎性和衰老相关疾病的发生、发展从而可能最终影响衰老的进程；Sirtl是一个哺乳动物NAD+依赖的脱乙酰基酶,在细胞周期停滞和DNA修复中起作用,其能减弱凋亡,调节细胞老化；p16相关于端粒缩短,在细胞衰老时其表达常明显增强；Klotho能影响细胞内信号通路,调节成纤维细胞生长因子23信号,p53/p21, cAMP,蛋白激酶C (PKC)和Wnt信号通路,修复内皮的机能障碍和调节一氧化氮的产生,从而可能影响衰老的进程。本研究采用候选基因和病例对照的方法,以中国沈阳地区汉族健康人群为研究对象,按照生物学年龄分组,用Snapshot方法检测Sirt1、IL-6、P16及Klotho四个基因10个位点的基因型及基因频率,探讨这些基因SNP与衰老的关系。所获得的结果有助于深入阐明衰老的分子遗传学机制,并可为实现在基因水平上的衰老个体化评价,乃至将来诊断治疗病理性衰老奠定重要的实验基础。材料与方法本研究采用病例对照研究,从1500余人中筛选出健康人442人,从反映脑功能、心功能、血管功能、肺功能、肾脏功能和反应日常生活状况的一般指标总计70个指标利用统计学方法挑选出生物学标志物并构建生物学年龄积分(Biological age score,BAS)计算公式,将研究对象按照生物学年龄分组,分为衰老组228例和年轻组213人。利用生物信息学方法在四个候选基因上选择10个SNPs遗传标记。利用Snapshot方法检测个体基因型。用拟合优度卡方检验验证每个SNP基因型在抽样群体中的分布是否符合H-W(Hardy*Weinberg)平衡,用Genepop3.4软件检验H-W平衡。采用SPSS统计软件分析每个SNP位点等位基因及基因型在年轻组与衰老组之间的分布是否有显著性差异,从而推测SNPs位点与衰老的关系。使用SPSS统计软件对相关数据进行X2检验、计量资料采用均数±标准差表示,以p<0.05为显著性水准。使用Shesis软件分析SNPs之间的连锁不平衡程度、分析同一基因不同位点之间的相互作用。实验结果1、挑选出7个生物学标志物TMT,CCR,MVES,IMTmin,Dmax,MMEF75/25, PP,并成功构建生物学年龄积分公式,BAS=-0.169(CCR)-0.179(MVES)+0.184 (IMTmin)+0.155(Dmax)-0.158(MMEF75/25)+0.226(CA)+0.148(PP)+0.159 (TMT).2、衰老组和年轻组每个SNP基因型的H-W平衡检验都符合H-W遗传平衡,我们所选择的研究对象的基因频率能够代表群体的基因分布。3、IL-6的基因多态性在健康衰老组和对照年轻组中频率分布比较：rs2066992位点两组间各基因型无显著性差异,X2=0.05,P=0.82；等位基因频率在两组间无显著性差异,X2=0.09,OR=0.95,95％CI:0.70-1.31,P=0.76:rs1524107位点两组间各基因型无显著性差异,X2=0.32,P=0.57；等位基因频率在两组间无显著性差异,X2=0.20,P=0.66,OR=0.932,95％CI:0.68-1.27.4、P16基因的基因多态性在衰老组和年轻组中频率分布比较：rs2811708位点两组间各基因型无显著性差异,X2=0.28,P=0.59；等位基因频率在两组间无显著性差异,X2=0.24,P=0.62, OR=0.94,95%CI:0.72-1.22; rs3731245位点两组间各基因型无显著性差异,X2=0.34,P=0.79；等位基因频率在两组间无显著性差异,X2=0.15, OR=0.94,95%CI=0.70-1.27, P=0.70。5、Klotho基因的基因多态性在衰老组和年轻组中频率分布比较：rs571118位点衰老组CC型频率高于对照组,而CT型和TT型频率低于对照组,有显著性差异X2=7.28,P=0.006；T等位基因和C等位基因在两组间有显著性差异,X2=6.78, P=0.009,OR=1.424,95%CI:1.09-1.86。rs1207568位点衰老组CC型,CT型和TT型频率在衰老组和年轻组比较,差异有显著性,X2=5.18,P=0.023；等位基因频率在两组间比较,也有显著性差异,X2=5.18, OR=1.31,95%CI=0.78-1.38, P=0.02。rs2149860位点两组间各基因型无显著性差异,X2=0.30,P=0.58；等位基因频率在两组间无显著性差异,X2=0.31, OR=1.08,95%CI=0.83-1.40, P=0.58。Klotho基因的rs648202位点两组间各基因型无显著性差异,X2=0.11,P=0.74；等位基因频率在两组间无显著性差异,X2=0.21, P=0.64, OR=0.93,95%CI:0.69-1.26。6、Sirt1基因的基因多态性在衰老组和年轻组中频率分布,Sirt1基因的rs3758391位点两组间各基因型无显著性差异,X2=0.13,P=0.71；等位基因频率在两组间无显著性差异,X2=0.71, OR=1.14,95%CI=0.83-2.02, P=0.02。Sirt1基因的rs4746720位点衰老与对照组的基因型及基因频率相比较,衰老组CC型频率低于对照组差异有显著意义：X2=3.85,P=0.04；C等位基因频率低于对照组,差异有显著性意义：X2=4.35, OR=1.33,95%CI=1.02-1.73, P=0.03。7、连锁不平衡分析结果Klotho的rs571118, rs2149860, rs 1207568, rs648202的四个位点之间显示D’<0.6, rs571118, rs2149860, rs 1207568, rs648202四个位点之间不存在连锁不平衡。IL-6的rs2066992, rs1524107之间D’=0.99, rs2066992, rs1524107位点之间强相关。P16的rs2811708, rs3731245的之间D’=0.92, rs2811708, rs3731245位点之间强相关。Sirt1的rs3758391, rs4746720位点之间进行连锁不平衡及单倍型分析。结果显不D’=0.53, rs3758391,rs4746720位点之间不相关。结论1、成功地构建了生物学年龄积分公式；按照生物学年龄分组,较传统时序年龄更合理地反映个体的衰老化进程。2、Klotho基因的rs571118的GG基因型和rs1207568位点CC基因型可能为沈阳汉族健康人群衰老的危险因素,其遗传易感性主要来自于等位基因G和C。Klotho基因的rs2149860和rs648202的两个位点的多态性和沈阳汉族健康人群的衰老未见相关。3、IL-6基因的rs2066992, rs1524107的两个位点的多态性和沈阳汉族健康人群衰老未见相关。4、P16基因的rs2811708, rs3731245的两个位点的多态性和沈阳汉族健康人群衰老未见相关。5、Sirtl基因的rs4746720位点CC基因型可能为沈阳汉族人群衰老的危险因素,其遗传易感性主要来自于等位基因C, Sirtl基因的rs3758391位点的多态性和沈阳汉族健康人群衰老未见相关。更多还原

【Abstract】 BackgroundAging also known as senescence, usually refers to the normal conditions of biological maturity, increased with age, their function decline, stable capacity and stress within the environment reduced capacity, structure, composition degeneration gradually towards death and irreversible. In the late 20th century, the theory of molecular biology and cell biology technology utilize in Gerontology research, the researchers found that although there has a wide range of the theories of aging, but no more than genetic and environmental aspects. Mutation of the WRN gene located on chromosome 8p-12 induce Wemer progeria syndrome (adult progeria); the same aging of different individuals have different phenotypes and different life; family investigation, twin, and popular disease studies all shown that aging is a highly individualized process, closely related to genetic factors.Rate of aging varies in different Individuals, the differences between human individuals has also increased with age. Some people are very good of physical and cognitive function in 85 years, but some people may occur a wide range of cognitive impairment or physical dysfunction in 55 years, Chronological age (CA) can not serve as aging the process of accurate instructions. For basic and clinical research of aging purposes, researchers concerned with aging changes in the individual the purpose of screening individuals at high risk of aging and provide timely intervention. To solve the above problems, researchers proposed the "Biological Age (BA)" concept. Biological age relative to the timing age of the individual functional state of their peers based on the evaluation of the individual’s functional status. Individual aging at different rates leading to timing differences in age and biological age, so the timing of any given age, individuals between the value of biological age may be different, and ultimately the individual can be expected with the aging process results in time and/or the magnitude of the difference was consistent.Decreased in senescent human body functions, and enhanced susceptibility to some diseases, some people will live longer seen as a successful aging phenotypes, from this point of view, some of the older disease-related genes and longevity-related genes can also be seen as aging-related genes. Many scholars believe that aging like hypertension, atherosclerosis, dementia, cancer and other disease, not the decision of a single gene, but is likely to be influenced by a large and complex aging-related genes in gene cluster. Study the aging process of gene expression profile, screening of new gene, isolated and cloned senescence-related genes, to understand its function, regulation, impact factor is the real way to find out the mechanism of aging. Biological function to genes as candidate genes for association studies of genetic susceptibility to the current aging of the main strategy. The results show that variations in the genome sequence, that gene polymorphism is to determine the susceptibility of the human body to age related diseases and resistance to aging and the clinical phenotype diversity of human response to drug treatment an important factor in differences.Polymorphism of DNA can be divided into three phases:Restriction Fragment Length Polymorphism, Microsatellite Polymorphisms and Single Nucleotide Polymorphisms (SNP). SNPs compared to the previous two generations its high density of genetic markers, genetic stability, representative, are distributed unevenly and its analysis automation.SNPs changes, especially in the coding region and the SNPs in the expression and regulation changes in the sequence, protein expression may lead to changes in the quality or quantity, thereby affecting protein structure or expression. Thus SNPs may help explain the phenotypic differences between individuals, groups and individual susceptibility to aging and longevity difference. Find the aging and diseases of aging-related SNP, is one of the important way to reveal the cause of human aging. Insulin/insulin like growth factor-1 (insulin/IGF-1) way to many kinds of protein interactions at the same time regulating the expression of a large number of genes, Sirt1, IL-6, P16, and Klotho genes or the involvement of four insulin/IGF-1 signaling pathway and the insulin/IGF-1 signaling pathway, or interactions which may affect the aging process. IL-6 is an inflammatory cytokine, which can affect the number of inflammatory and aging-related diseases, the development of which may ultimately affect the aging process; Sirt1 is a mammalian NAD+dependent deacetylase, in the cell cycle arrest and play a role in DNA repair, which can be reduced apoptosis, regulation of cell aging; p16 related to telomere shortening, cell senescence in markedly enhanced when the regular expression; Klotho can affect the intracellular signaling pathway to regulate fibroblast growth factor 23 signals, p53/p21, cAMP, protein kinase C (PKC) and the Wnt signaling pathway, repair, and regulation of endothelial dysfunction, production of nitric oxide, which may affect the aging process.In this study, case-control candidate gene approach to China, Shenyang Han population, according to biological age groups, using Snapshot method detected 10 SNPs in Sirtl, IL6, P16, and Klotho genes, and detect the genotypes and gene frequency of these genes the relationship between these SNPs and aging. The results obtained will help to further elucidate the molecular genetic mechanisms of aging, and can achieve the level of the genes on the aging individual evaluation, as well as the future treatment of pathological aging diagnostic lay an important experimental basis.Materials and methodsThis study used case-control study, selected from more than 1,507 people, choose 442 healthy people, the 70 indexes reflecting the brain functions, heart function, vascular function, lung function, kidney function and reaction conditions of the general indicators of daily using statistical methods construction the Biological age score (BAS) formula, the study according to biological age group, divided into 228 patients aged group and 213 young people. Bioinformatics approach to select 10 SNPs in four of candidate genes. Individual genotypes were detected using Snapshot. With the goodness of fit chi-square test verifies that each SNP genotype distribution in the sample population is consistent with HW equilibrium, using Genepop3.4 software testing H-W (Hardy-Weinberg) equilibrium. Using SPSS statistical software to analyze each SNP allele and genotype in the young group and the distribution between the aging group is significantly different to speculate SNPs, the relationship with aging. Using SPSS statistical software-related data X2 test, measurement data with mean±sd, p<0.05 as significant difference. Shesis software analysis using linkage disequilibrium between SNPs level of the same gene interaction between the different sites.Results1. Selected seven biomarkers TMT, CCR, MVES, IMTmin, Dmax, MMEF75/25, PP, and biological age successfully constructed integral formula, BAS=-0.169 (CCR)-0.179(MVES)+0.184 (IMTmin)+0.155 (Dmax)-0.158 (MMEF75/25)+0.226 (CA) +0.148 (PP)+0.159 (TMT).2. Aging and young groups for each SNP genotype H-W equilibrium test are keep balance, we chose the study sample can represent the whole people.3. The frequency distribution of IL-6 gene polymorphism in healthy young and aging group compared:rs2066992 locus genotypes between the two groups no significant difference, X2=0.05, P=0.82; alleles between the two groups no significant difference, X2=0.09, OR=0.95,95%CI=0.69-1.30, P=0.76; rs1524107 locus genotypes between the two groups no significant difference, X2=0.32, P=0.57; allele frequencies between the two groups no significant difference, X2=0.20, P=0.66, OR=0.932,95% CI:0.685-1.268.4. The frequency distribution of P16 gene polymorphism in aging and young groups compared:rs2811708 locus genotypes between the two groups no significant difference, X2=0.28, P=0.59; allele frequencies between the two groups There was no significant difference, X2=0.24, P=0.62, OR=0.94,95%CI:0.718-1.219; rs3731245 locus genotypes between the two groups no significant difference, X2=0.34, P=0.79; allele frequency between the two groups have no significant difference, X2=0.15, OR=0.94,95%CI= 0.70-1.27, P=0.70.5. The frequency distribution of Klotho gene polymorphism in aging and young groups compared:rs571118 site CC genotype frequency higher aging group, while the CT type and TT genotype frequency lower than the control group, there was significant difference in X2=7.28, P=0.006; T allele and C allele has significant difference between the two groups, X2=6.78, P=0.01, OR=1.42,95%CI:1.09-1.86. rs1207568 CC-type locus aging group, CT-based and TT genotype frequency in the aging and young groups, the difference was significant, X2=5.18, P=0.02; there are significant differences allele frequencies between the two groups, X2=5.18, OR=1.308,95%CI=0.78-1.38, P=0.02. rs2149860 genotypes between the two groups have no significant difference, X2=0.30, P=0.58; allele frequencies between the two groups no significant difference, X2=0.31, OR=1.077,95%CI=0.827-1.403, P=0.58. Klotho gene rs648202 genotypes between the two groups have no significant difference, X=0.11, P=0.74; allele frequencies between the two groups have no significant difference, X=0.21, P=0.64, OR=0.93, 95%CI:0.69-1.26.6. Sirt1 gene polymorphism in aging and young groups in the frequency distribution, Sirtl gene locus rs3758391 genotype between the two groups no significant difference, X2=0.13, P=0.71; allele frequencies in two there were no significant differences, X2=0.71, OR=1.14,95%CI=0.83-2.02, P=0.026. Sirtl gene rs4746720 genotype CC genotype frequency aging group compared with the control group showed significant:X2=3.85, P=0.04; C lower than the control group allele frequency The difference was statistically significant:X2=4.35, OR=1.328,95%CI= 1.02-1.73, P= 0.03.7. Linkage Disequilibrium analysisRs571118, rs2149860, rs 1207568, rs648202 in Klotho, the four sites D′<0.6, rs571118, rs2149860, rs1207568, rs648202 has no correlation with each other.The D′of rs2066992, rs1 524107 in IL-6=0.99, rs2066992 strong correlation with rs1524107. The D’of rs2811708, rs3731245 in P16=0.92, rs2811708 strong correlation with rs3731245.Rs3758391, rs4746720 of Sirtl gene D’=0.53. Rs3758391and rs4746720 has no relation.Conclusion1. Biological age score formula successfully constructed, devide group using biological age, more reasonable than the traditional timing age of the individual to reflect the aging process.2. Klotho gene rs571118 of the GG genotype and rs1207568 CC genotype may be sites of Shenyang Han population aging risk factors, mainly from the genetic susceptibility alleles G and C. Klotho gene rs2149860 and rs648202 no correlation with Shenyang Han population aging.3. IL-6 gene rs2066992, rs 1524107 polymorphism has no correlation with Shenyang Han population aging.4. P16 gene rs2811708, rs3731245 polymorphism as no correlation with Shenyang Han population aging.5. Sirtl gene rs4746720 CC genotype may impact as Shenyang Han population aging risk factors, mainly from the genetic susceptibility allele C, Sirtl gene rs3758391 polymorphism and Shenyang Han population aging no correlation.更多还原