新生儿端粒长度的影响因素研究

发布时间：2018-04-11 05:46

本文选题：脐血 + 端粒长度　；参考：《南京医科大学》2016年博士论文

【摘要】：端粒(Telomere)是特异结合蛋白与真核细胞染色体末端的TTAGGG六个碱基重复序列的复合体,在维持染色体的完整性和基因组稳定性方面具有极其重要的作用。随着细胞分裂,端粒进行性缩短,端粒长度可以反映机体在细胞水平的衰老程度,目前许多研究证实成人端粒长度可以作为年龄相关疾病的风险标志物。虽然组织来源的细胞能够为研究端粒长度与疾病关系提供更直接的证据,但由于不同组织间端粒长度存在高度的相关性,外周血样本产生DNA质量高且更加容易获取,因此目前外周血被广泛用于端粒长度的研究中。流行病学研究发现成人的端粒长度主要受到环境因素和遗传因素的共同影响,端粒长度取决于出生时端粒长度及出生后氧化应激程度。不同个体出生时端粒长度有明显差异,且研究发现生命早期端粒长度比生命后期端粒长度能够更好地预测寿命并且能对个体产生终身的影响,其影响程度超过生命后期的其他风险暴露,因此研究新生儿端粒长度具有重要的意义。但目前新生儿端粒长度的研究样本量较小,影响因素不确切,研究结果也不一致。另外研究证实新生儿端粒长度与遗传因素相关,但目前国内外仅有研究成人端粒长度的遗传变异,新生儿端粒长度与遗传变异的研究尚未见报道。因此,本研究在第一部分主要探索环境因素对新生儿端粒长度的影响;第二部分主要研究遗传因素对新生儿端粒长度的影响。第一部分:环境因素对新生儿端粒长度的影响流行病学研究已经对新生儿端粒长度的社会人口学因素及环境暴露因素进行了探索,Friedrich等对15例新生儿的研究发现,孕周、新生儿出生体重等都影响脐血端粒长度,Entringer等人对27例新生儿的研究发现,孕期的精神压力也显著影响脐血端粒长度,而Cross JA等在81例新生儿中的研究发现,脐血白细胞端粒长度与母亲年龄、孕周、吸烟、新生儿出生体重等均无明显相关关系。因此,目前已有的研究不仅样本量较小,而且研究结论尚不一致。本研究采用横断面研究深入分析影响新生儿脐静脉血相对端粒长度的相关环境因素。研究对象为南京鼓楼医院出生的444例新生儿,对新生儿母亲进行问卷调查、体格检查和临床相关指标检测,采集新生儿脐静脉血及与其配对的母体外周血样本。采用定量PCR方法用特异性引物将待测基因组的端粒(Telomere,T)序列和单拷贝基因(Single copy gene,S)序列同时进行扩增,用二者拷贝数的比值(T/S)来定量相对端粒长度(Relative telomere length,RTL)。为保证端粒长度检测的准确性,实验操作中进行复孔平行设置和重复检测等质量控制措施。采用配对T检验进行母儿端粒长度之间的比较,采用方差分析和t检验进行不同组间相对端粒长度差异的比较;采用一般线性模型分析新生儿端粒长度与母亲端粒长度等连续性变量之间的相关性。研究结果显示,新生儿端粒长度(均数为0.358)显著大于母亲端粒长度(均数为0.309)(P0.001),未发现母儿端粒长度之间有明显相关关系(R2 = 0.004,P=0.174)。母亲受教育程度越高,新生儿端粒长度越长(P=0.022)。孕周、分娩方式、新生儿性别、出生体重与母儿端粒长度均未见明显关联(P0.05)。孕期摄入奶制品较多,母亲端粒长度较短(P=0.048),孕期蔬菜水果、肉制品、动物内脏、鱼虾、豆制品、煎烤及腌制食品、叶酸以及多种维生素摄入不同组之间,新生儿和母亲端粒长度均未发现显著差异(P0.05)。孕期饮用咖啡新生儿端粒长度短(P=0.011)。孕期二手烟暴露、饮酒、饮茶情况对母亲和新生儿端粒长度未见明显影响(P0.05)。孕期经常参加体育锻炼的孕妇端粒长度长于不锻炼者或者很少锻炼者(P=0.01),而新生儿端粒长度未见显著差异(P=0.187)。本研究较全面评估了孕期的重要环境因素对孕妇及新生儿端粒长度的影响,研究结果将为揭示端粒在疾病发生中的作用提供了重要线索。第二部分:遗传因素对新生儿端粒长度的影响研究表明,遗传因素能够解释36%～90%的个体之间端粒长度的变异,因此,研究影响端粒长度的遗传变异位点和基因对于阐明端粒长度受遗传调控的内在机制具有十分重要的意义。全基因组关联研究(Genome-wide association study,GWAS)方法近年被广泛应用于基因与疾病的关联研究,其通过对大规模的群体DNA样本进行全基因组高密度遗传标记分型,从而寻找与复杂疾病相关的遗传因素的研究方法,全面揭示疾病发生、发展与治疗相关的遗传基因。目前为止,基于欧洲人群的GWAS研究发现有多个遗传变异位点与端粒长度相关,其中包括3q26(TERC)、4q32.2(NAF1)、5p15.33(TERT)、10q24.33(OBFC1)和 20q13.3(RTEL1)等这些已经被研究证明直接参与端粒生物学功能调控的基因的染色体区域[37-41],也包含2p16.2(ACYP2)和19p12(ZNF208)等还没有相关证据证明其参与端粒生物学调控的基因的染色体区域。这些研究发现为阐明影响端粒长度的遗传机制提供重要线索。但是,这些研究均基于成年人,其端粒长度既与生命早期的端粒长度有关,也受到生长过程中环境暴露导致的端粒缩短速度影响。然而,目前关于新生儿端粒长度相关遗传变异的研究尚未见报道。本研究利用TaqMan 7900基因分型平台在444例脐静脉血及与其对应的母血中对目前GWAS已报道端粒长度相关位点进行研究。为了更加全面的分析既往GWAS报道端粒长度相关遗传变异与母儿端粒长度的相关性,本研究对这8个遗传变异位点的基因型进行加权遗传评分(Weighted genetic score,WGS)的计算,分析WGS与母体外周血及脐静脉血端粒长度的相关关系,评价8个遗传变异与母儿端粒长度的综合效应。遗传变异及加权遗传评分与端粒长度的相关性采用一般线性模型分析。研究结果显示,欧洲人群端粒长度显著相关的遗传变异rs10936599和rs2736100与中国孕妇端粒长度显著相关(β=0.0119,P=00.41及β=0.011,P=0.046),已知的8个遗传变异的加权效应与母亲端粒长度的相关性更强(R2=0.012,P=0.025);本研究发现,上述8个遗传变异单独或联合与新生儿端粒长度均不存在显著关联(P=0.716)。本研究首次发现已知端粒长度相关的遗传变异可能不影响新生儿的端粒长度,但影响成长过程中不同个体对环境暴露导致端粒缩短的易感程度。本研究首次分析了遗传变异与新生儿及与孕母端粒长度的相关关系,为探索端粒长度遗传调控机制研究提供了重要线索。
[Abstract]:Telomere length (Telomere) is a complex of specific binding protein and the ends of eukaryotic chromosomes TTAGGG six nucleotide repeats, plays an important role in maintaining chromosome integrity and genomic stability. With cell division, telomere shortening of telomere length, can reflect the aging degree of the body at the cellular level, at present a lot of research that telomere length can be used as a marker of risk of adult age related diseases. Although the cells can provide more direct evidence for the research on the relationship between telomere length and disease, but due to the different interstitial telomere length has high correlation with peripheral blood samples of DNA have high quality and more easily accessible, so the peripheral blood was widely used in the study of telomere length in adult. Epidemiological studies have found that telomere length is mainly affected by environmental factors and genetic factors. The combined effect on telomere length in telomere length and after birth when the degree of oxidative stress. Different individuals born when telomere length were significantly different, and the study found that early life telomere length of telomere length in later life to be able to better predict the life and production of individual lifelong effects, the effects of other risk more than later in life so the study of neonatal exposure, telomere length has important significance. But at present the research sample of neonatal telomere length is smaller, the influence factors is not exact, the results are not consistent. Other studies confirmed neonatal telomere length and genetic factors related to genetic variation, but the research at home and abroad only adult telomere length and neonatal telomere length and genetic variation has not been reported. Therefore, this study in the first part mainly explores the environmental factors on neonatal telomere length The second part mainly studies the influence; influence of genetic factors on neonatal telomere length. The first part: the epidemiological study on the effects of environmental factors on neonatal telomere length has social and demographic factors on neonatal telomere length and environmental exposure factors were explored, a study of 15 cases of neonatal Friedrich, gestational age, birth weight and so on telomere length of umbilical cord blood of 27 neonates, Entringer et al found that pregnancy stress has a significant influence on telomere length and umbilical cord blood, in 81 cases of neonatal Cross JA found that umbilical cord blood leukocyte telomere length and maternal age, gestational age, smoking, there was no obvious correlation between the birth weight of newborns. Therefore, the present study not only the small sample, but the conclusion is not consistent. This study used a cross-sectional study in-depth analysis of the impact of neonatal umbilical The relative telomere length of venous blood related environmental factors. The research object for 444 neonates born in Nanjing Gulou Hospital, conducted a questionnaire survey of mothers, physical examination and detection of clinically relevant indicators, collecting umbilical cord blood and maternal matched peripheral blood samples. Using quantitative PCR method to test the genome by using specific primers of the telomere (Telomere, T) sequence and single copy gene (Single copy gene, S) sequence were amplified with two copy number ratio (T/S) to quantify the relative telomere length (Relative telomere, length, RTL). In order to ensure the accuracy of detection of telomere length, control measures and holes arranged in parallel repetition detection quality experimental operation. Paired T test was used for comparison between the telomere length of mother and infant, compared with analysis of variance and t test were different between group differences in the relative telomere length; The general linear model to analyze the correlation between neonatal telomere length and mother telomere length continuity variables. The results showed that the telomere length of newborn (mean 0.358) was significantly higher than the mother of telomere length (mean 0.309) (P0.001), there was no obvious correlation between maternal telomere length (R2 = 0.004. P=0.174). The higher the education degree of mother, neonatal telomere length (P=0.022). The longer the gestational age, mode of delivery, neonatal sex, birth weight and maternal telomere length had no significant correlation (P0.05). During the intake of dairy products more, mother short telomere length (P=0.048) during pregnancy, fruits and vegetables, meat products, animal offal, fish, beans, roasted and salted food, folic acid and vitamins intake between different groups, no difference was found between the newborn and mother telomere length (P0.05) during pregnancy. Drinking coffee (short telomere length in neonates P=0.011). Prenatal exposure to secondhand smoke, drinking tea, the effects on maternal and neonatal telomere length was not obvious (P0.05) during pregnancy. The pregnant women often take part in physical exercise is longer than the telomere length do not exercise or rarely exercise (P=0.01), and neonatal telomere length have no significant difference (P=0.187). This study is a comprehensive assessment of the impact the important environmental factors of pregnancy on maternal and neonatal telomere length, the results will reveal the role of telomeres in the disease provides an important clue. The second part: study the influence of genetic factors on neonatal telomere length showed that genetic factors could explain 36% to telomere length variation between 90% individuals so the effect of telomere the length of the genetic mutations and gene has very important significance to elucidate the mechanism underlying telomere length by genetic manipulation. A genome-wide association study (Geno Me-wide association study, GWAS) related research methods in recent years has been widely used in gene and disease, which based on the large-scale group DNA samples of whole genome of high density genetic marker type, in order to find out the research methods of genetic factors associated with complex diseases, fully reveal the disease occurrence, development and treatment of related genes. So far, GWAS of European populations found multiple genetic mutations associated with telomere length based on, including 3q26 (TERC), 4q32.2 (NAF1), 5p15.33 (TERT), 10q24.33 (OBFC1) and 20q13.3 (RTEL1) etc. These have been studied that chromosomal region [37-41] directly involved in telomere biology function gene also, including 2p16.2 (ACYP2) and 19p12 (ZNF208) is not related to evidence of chromosomal regions in telomere biology gene. These findings in order to elucidate the effect Provide important clues to the genetic mechanism of telomere length. However, these studies are based on the adults, the telomere length of telomere length and early life, but also by the growth process of environmental exposure leads to telomere shortening speed. However, the current research on the length of the genetic variants associated with neonatal telomere has not been reported. This study uses TaqMan 7900 genotyping platforms in 444 cases of umbilical cord blood and maternal blood in corresponding research on the current GWAS has been reported to related sites. Correlation between telomere length in order to more comprehensive analysis of the previous GWAS reports related to telomere length and the genetic variation of maternal telomere length, weighted score of the genetic genotype on the 8 the genetic mutation (Weighted genetic, score, WGS) calculations, analysis of the relationship between WGS and maternal peripheral blood and umbilical vein blood in telomere length and 8 genetic evaluation The comprehensive effect of variation and maternal telomere length. Genetic variation and genetic correlation weighted score and telomere length by using the general linear model analysis. The results showed that the correlation was significantly related to telomere length in European populations and genetic variation of rs10936599 and rs2736100 in pregnant women China telomere length (beta =0.0119, P=00.41 and beta =0.011, P=0.046), more relevant the 8 known genetic variation and mother weighting effect of telomere length (R2=0.012, P=0.025); the study found that the 8 genetic variants alone or in combination with the telomere length of newborns does not exist significant association (P=0.716). This is the first study found that genetic variation associated with telomere length may not be known to affect neonatal telomere length, but susceptible to different individual environmental exposure leads to telomere shortening of the growth process. This is the first study to analyze the genetic variability and neonatal and The correlation with the telomere length of pregnant women provides an important clue for the study of the genetic regulation mechanism of telomere length.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R722.1

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