同型半胱氨酸与神经管畸形的相关病因学研究进展

神经管畸形(neural tube defects,NTDs)是一种最常见的严重中枢神经系统先天性畸形,在世界各地均有发生。它是造成流产、死产的主要原因之一,即便胎儿存活,也严重影响患儿的生长发育和生活质量,同时给家庭和社会带来沉重的精神压力和经济负担。神经管畸形的发生绝大多数是由遗传因素与环境因素相互作用的结果,若孕妇在孕早期缺乏叶酸、高热、接触射线、服用药物、感染或发生妊娠期糖尿病等条件下,结合遗传因素作用,均有可能导致神经管畸形的发生,但其目前其的确切病因及其发病机制仍有待深入研究。大量研究表明,在孕妇血清中低叶酸、低维生素B12水平及高血浆同型半胱氨酸(Hcy)水平,都与NTDs的发生密切相关。本文主要围绕同型半胱氨酸的代谢,从分子水平和基因水平对与神经管畸形相关的各因素做一综述。     

叶酸与神经管畸形相关研究进展

神经管畸形(neural tube defects,NTDs)是胚胎发育过程中由于神经管闭合失败所导致的出生缺陷,也是新生儿最常见的先天畸形之一。神经管畸形的发生是一个多因素、多步骤的复杂过程。近年来,叶酸已逐渐成为NTDs研究中专注度最高的一个环境因素,但其在神经管闭合过程中的作用机制尚不完全清楚。本文就近年来叶酸在神经管发育中的作用机制作一综述,简要阐明叶酸转运机制、叶酸代谢、叶酸与DNA甲基化、叶酸与组蛋白修饰等方面在早期胚胎发育神经管形成过程中的作用。     

与神经管畸形相关的基因学研究进展

神经管畸形（neural tube defects,NTDs）是指由于在胚胎发育过程中,神经管闭合不全所引起的一组出生缺陷,包括无脑儿、脊柱裂、脑积水、脑或脑脊膜膨出等,其危害极大,严重影响患儿的生理发育和生活质量,给家庭和社会带来沉重的精神压力和经济负担。大多数研究认为神经管畸形是多因素多基因的遗传疾病,是遗传、环境、营养因素共同作用、交互影响的结果,目前还不能用一种单一原因解释该疾病的发生。本文主要从导致神经管畸形发生的叶酸代谢酶基因多态性、神经管形态学方面的相关基因研究等做一综述。     

ERK1/2在高温致神经管畸形神经上皮细胞中的表达变化

目的 探讨高温致神经管畸形(NTDs)作用的分子机制,为防治NTDs的发生提供理论依据.方法 在高温致金黄地鼠NTDs模型的基础上,应用免疫荧光染色技术,观察NTDs发生过程中p-ERK1/2在鼠胚神经上皮细胞中的表达变化.结果 对照组和实验组孕鼠在高温水浴处理后16、24h,p-ERK1/2免疫阳性产物分布于鼠胚神经上皮细胞和周围间充质细胞的胞浆中;水浴后36、60h,p-ERK1/2表达部位出现了由细胞浆向细胞核的转移;高温处理后,p-ERK1/2在实验组各期胚胎神经上皮细胞内的表达均比对照组减弱.结论 ERK1/2参与胚胎神经管的发育过程,其表达降低在高温致神经管畸形的发生中起重要作用.     

血小板源性生长因子受体α与高温致神经管畸形发生关系的研究

目的探讨PDGFR-α在高温致神经管畸形(NTDs)中的作用.方法在高温致神经管畸形动物模型上,采用免疫组织化学和图像分析技术,研究血小板源性生长因子受体α(PDGFR-α)在发育不同阶段的神经上皮中的表达,并观察高温对其表达的影响.结果在正常对照组,PDGFR-α广泛分布于神经管及其周围组织中;在高温致畸组,神经上皮中PDGFR-α的表达明显减弱,甚至不表达.结论 PDGFR-α的表达与神经管正常发育密切相关,其表达的减少可能是高温致NTDs机制中的重要环节.     

eNOS基因多态性与神经管畸形的研究进展

神经管畸形(neural tube defects,NTDs)是一类常见的出生缺陷,严重威胁着妇女儿童的身心健康和人口素质的提高,给社会的发展带来沉重负担,可引起孕妇流产、婴儿死亡和终生残疾。NTDs可分为无脑儿、脑膨出和脊柱裂三种类型,其病因和具体的发病机制尚不清楚。国内外多数研究认为,NTDs是一种由基因的多态性和环境因素所引起的严重的基因突变,还不能用一种单一原因来解释该病的发生。目前的研究热点是易感基因与NTDs的关系,内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)基因最近已被认为是导致NTDs发生的重要候选基因。eNOS基因的点突变或成串突变可以导致酶活性的变化,使eNOS的表达上调,引起NO分泌的异常,促进神经元的凋亡,进而导致大脑的发育异常。本文从eNOS基因多态性与NTDs的相关性研究进行综述。     

大脑皮层发育畸形及分子遗传机理研究进展

大脑皮层(cerebral cortex)发育是一个非常复杂的过程,主要包括神经干细胞的自我更新、分化、迁移和成熟等步骤。目前,已知多种因素可影响大脑皮层的正常发育并导致畸形的发生,并且随着产妇平均孕龄的不断增高和食品及环境因素的改变,大脑发育畸形的发病率正不断增加。充分了解大脑正常发育的分子机理和各种皮层畸形的发病机制对于人类相关疾病的早期诊断和治疗及优生优育都极为重要。该文首先简单总结了可能参与调控大脑皮层发育过程的多条信号通路,然后阐述了八种常见的人类皮层发育畸形的基本临床特征和分子遗传机理方面的研究进展,以期为今后相关领域的研究提供一些有用的参考信息。     

维甲酸致小鼠胚胎畸形发生过程中维甲酸受体α/β及β-catenin和caspase-3基因的表达变化

流行病学研究显示,在胚胎发育过程中摄入过多维甲酸可致各种发育缺陷,其中神经管畸形最为常见. 因此有必要探明维甲酸致各种发育缺陷的发生机制,以便为各种生长缺陷的预防和治疗提供实验依据. 用 RT-PCR 及蛋白质印迹技术,探测了过量维甲酸对昆明小鼠胚胎神经管中维甲酸受体α/β及β-catenin 和 caspase-3 基因表达的调整. 结果显示,在神经管闭合期过量维甲酸显著降低了维甲酸受体α/β及β-catenin 和 caspase-3 的基因表达,神经管闭合后,维甲酸受体β、β-catenin 及 caspase-3 的基因表达又出现了一个明显的回升过程. 提示,过量维甲酸改变了昆明小鼠胚胎神经管中维甲酸受体α/β及β-catenin 和 caspase-3 基因的正常时间表达模式,这种异常的基因表达模式可能参与了维甲酸致昆明小鼠胚胎畸形的发生机制.     

转化生长因子beta在热应激导致小鼠神经管畸形发生中的表达

本研究旨在探讨热应激致神经管畸形（NTDs）中转化生长因子beta（TGF-beta）的表达情况。选用昆明小鼠40只,实验组20只小鼠于妊娠后8．5天（E8．5）在42℃温箱中喂养30分钟,进行热应激处理,建立小鼠胚胎NTDs模型。对照组不处理。11．5d取胎鼠,通过体视显微镜和组织学切片观察实验组和对照组神经管发育情况,计算神经管畸形发生率;     

丙戊酸在神经发育中的作用

丙戊酸(valproic acid,VPA)是一种分支短链脂肪酸,是一种抗癫痫药物,主要用于癫痫,但也用于双相情感障碍和偏头痛,具有一定的神经保护作用。然而,产前暴露于丙戊酸会增加儿童患先天性畸形、孤独症和神经管缺陷的风险,对子代的神经发育有一定的毒性作用。但丙戊酸具体的致病机制目前还不清楚。目前多项研究已经发现丙戊酸会对子代的不同脑区造成影响,致使脑区多个通路发生改变。本文综述了目前关于丙戊酸对神经发育影响的研究进展,探讨了丙戊酸的潜在致病机制和相关因素。     

Temporal and Spatial Analysis of Neural Tube Defects and Detection of Geographical Factors in Shanxi Province,China

BackgroundNeural tube defects (NTDs) are congenital birth defects that occur in the central nervous system, and they have the highest incidence among all birth defects. Shanxi Province in China has the world’s highest rate of NTDs. Since the 1990s, China’s government has worked on many birth defect prevention programs to reduce the occurrence of NTDs, such as pregnancy planning, health education, genetic counseling, antenatal ultrasonography and serological screening. However, the rate of NTDs in Shanxi Province is still higher than the world’s average morbidity rate after intervention. In addition, Shanxi Province has abundant coal reserves, and is the largest coal production province in China. The objectives of this study are to determine the temporal and spatial variation of the NTD rate in rural areas of Shanxi Province, China, and identify geographical environmental factors that were associated with NTDs in the risk area.MethodsIn this study, Heshun County and Yuanping County in Shanxi Province, which have high incidence of NTDs, were selected as the study areas. Two paired sample T test was used to analyze the changes in the risk of NTDs from the time dimension. Ripley’s k function and spatial filtering were combined with geographic information system (GIS) software to study the changes in the risk of NTDs from the spatial dimension. In addition, geographical detectors were used to identify the risk geographical environmental factors of NTDs in the study areas, especially the areas close to the coal sites and main roads.ResultsIn both Heshun County and Yuanping County, the incidence of NTDs was significantly (P<0.05) reduced after intervention. The results from spatial analysis showed that significant spatial heterogeneity existed in both counties. NTD clusters were still identified in areas close to coal sites and main roads after interventions. This study also revealed that the elevation, fault and soil types always had a larger influence on the incidence of NTDs in our study areas. In addition, distance to the river was a risk factor of NTDs in areas close to the coal sites and main roads.ConclusionThe existing interventions may have played an important role to reduce the incidence of NTDs. However, there is still spatial heterogeneity in both counties after using the traditional intervention methods. The government needs to take more measures to strengthen the environmental restoration to prevent the occurrence of NTDs, especially those areas close to coal sites and main roads. The outcome of this research provides an important theoretical basis and technical support for the government to prevent the occurrence of NTDs.     

The emerging role of epigenetic mechanisms in the etiology of neural tube defects

The molecular requirements for neural tube closure are complex. This is illustrated by the occurrence of neural tube defects (NTDs) in many genetic mouse mutants, which implicate a variety of genes, pathways and cellular functions. NTDs are also prevalent birth defects in humans, affecting around 1 per 1,000 pregnancies worldwide. In humans the causation is thought to involve the interplay of fetal genes and the effect of environmental factors. Recent studies on the etiology of human NTDs, as well as analysis of mouse models, have raised the question of the possible involvement of epigenetic factors in determining susceptibility. A consideration of potential causative factors in human NTDs must now include both alterations in the regulation of gene expression, through mutation of promoter or regulatory elements and the additional analysis of epigenetic regulation. Alterations in the epigenetic status can be directly modified by various environmental insults or maternal dietary factors.Key words: neural tube defects, diet, folic acid, epigenome, epigenetic regulation, methylation, chromatin, histones, acetylation     

The emerging role of epigenetic mechanisms in the etiology of neural tube defects

The molecular requirements for neural tube closure are complex. This is illustrated by the occurrence of neural tube defects (NTDs) in many genetic mouse mutants, which implicate a variety of genes, pathways and cellular functions. NTDs are also prevalent birth defects in humans, affecting around 1 per 1000 pregnancies worldwide. In humans the causation is thought to involve the interplay of fetal genes and the effect of environmental factors. Recent studies on the aetiology of human NTDs, as well as analysis of mouse models, have raised the question of the possible involvement of epigenetic factors in determining susceptibility. A consideration of potential causative factors in human NTDs must now include both alterations in the regulation of gene expression, through mutation of promoter or regulatory elements, and the additional analysis of epigenetic regulation. Alterations in the epigenetic status can be directly modified by various environmental insults or maternal dietary factors.     

Higher serum homocysteine and lower thyroid hormone levels in pregnant women are associated with neural tube defects

BackgroundThis study tested the hypothesis that abnormal maternal metabolism of both homocysteine and thyroid hormone network in pregnant women is associated with neural tube defects (NTDs) in a part of China with high NTD prevalence.MethodsA case–control study was performed between 2007 and 2009 in Lüliang Mountains, Shanxi Province. This study included 83 pregnant women who had fetuses with NTDs (cases) and 90 pregnant women with normal fetuses (controls). In addition, a cell model to illustrate the epidemiological findings was established.ResultsFetuses of mother who had both high total homocysteine (tHcy) and inadequate free thyroxine were 3 times more at risk of developing NTDs (adjusted odds ratio = 3.5; 95 % confidence interval = 1.2–10.4; cases vs. controls) using multivariate logistic regression models. Furthermore, biological interaction between metabolisms of Hcy and thyroid hormones was demonstrated in vitro. In homocysteine thiolactone of a metabolite of Hcy-treated mouse embryonic neural stem NE4C cells, genes (Bmp7, Ctnnb1, Notch 1, Gli2, and Rxra) related to both neural tube closure and thyroid hormone network were shown to be regulated by H3K79 homocysteinylation, which increased their expression levels.ConclusionsThe effect of maternal serum high tHcy on risk of developing NTDs is depended on maternal serum level of thyroxine. Meanwhile, a higher level of tHcy might also affect both maternal metabolism of thyroid hormone and neural tube closure in embryogenesis through homocysteinylation of histones.     

碱基切除修复基因Lig3对小鼠胚胎神经发育的影响

摘要 目的：DNA连接酶III（DNA ligase III, Lig3）基因是碱基切除修复通路中的关键基因,在胚胎发育过程中发挥重要作用,通过研究Lig3基因在叶酸代谢障碍状态下的表达情况,探讨其对小鼠胚胎神经发育的影响。方法：采用无特定病原体（specific pathogen free, SPF）级C57BL/6J成年小鼠（8-9周,18-20 g）,雌雄1:1合笼,孕鼠随机分为实验组和对照组,孕7.5天实验组腹腔注射4.5 mg/kg体重甲氨蝶呤（Methotrexate, MTX,二氢叶酸还原酶抑制剂）诱导产生叶酸代谢障碍的小鼠神经管畸形（neural tube defects, NTDs）模型,对照组腹腔注射等体积的生理盐水。孕10.5天体视显微镜下观察胎鼠的发育情况。同时利用200 nM的MTX建立叶酸代谢障碍的小鼠神经干细胞模型。在模型建立成功的基础上,应用实时荧光定量聚合酶链反应(Real time quantitative PCR,RT-qPCR)及免疫印迹（Western blot）等方法研究碱基切除修复通路相关基因Lig3的表达水平。结果：4.5 mg/kg 体重MTX处理孕鼠后胎鼠NTDs的发生率为31.1%（19/61）,而正常对照组未见胎鼠NTDs的发生。在体视显微镜下可见NTDs胎鼠神经管未闭合,而正常胎鼠发育完好。RT-qPCR检测发现叶酸代谢障碍小鼠NTDs 胚胎神经组织中Lig3 mRNA的表达水平明显低于对照组（P<0.05）。Western blot检测发现,与对照组相比,叶酸代谢障碍NTDs胎鼠神经组织中Lig3蛋白水平明显降低（P<0.05）。同时,在MTX处理的神经干细胞中,Lig3的表达水平明显低于对照组（P<0.05）。对凋亡相关蛋白Cleaved caspase-3进行检测发现MTX处理后的NTDs胎鼠神经组织及细胞模型中其表达均明显增加,表明细胞凋亡增加。结论：在叶酸代谢障碍前提下,Lig3表达降低,DNA修复功能减弱,细胞凋亡增加,导致NTDs的发生,为NTDs及出生缺陷的防控提供新思路。     

Mouse as a model for multifactorial inheritance of neural tube defects

Neural tube defects (NTDs) such as spina bifida and anencephaly are some of the most common structural birth defects found in humans. These defects occur due to failures of neurulation, a process where the flat neural plate rolls into a tube. In spite of their prevalence, the causes of NTDs are poorly understood. The multifactorial threshold model best describes the pattern of inheritance of NTDs where multiple undefined gene variants interact with environmental factors to cause an NTD. To date, mouse models have implicated a multitude of genes as required for neurulation, providing a mechanistic understanding of the cellular and molecular pathways that control neurulation. However, the majority of these mouse models exhibit NTDs with a Mendelian pattern of inheritance. Still, many examples of multifactorial inheritance have been demonstrated in mouse models of NTDs. These include null and hypomorphic alleles of neurulation genes that interact in a complex fashion with other genetic mutations or environmental factors to cause NTDs. These models have implicated several genes and pathways for testing as candidates for the genetic basis of NTDs in humans, resulting in identification of putative pathogenic mutations in some patients. Mouse models also provide an experimental paradigm to gain a mechanistic understanding of the environmental factors that influence NTD occurrence, such as folic acid and maternal diabetes, and have led to the discovery of additional preventative nutritional supplements such as inositol. This review provides examples of how multifactorial inheritance of NTDs can be modeled in the mouse. Birth Defects Research (Part C) 96:193–205, 2012. © 2012 Wiley Periodicals, Inc.     

Mouse mutants with neural tube closure defects and their role in understanding human neural tube defects

BACKGROUND: The number of mouse mutants and strains with neural tube closure defects (NTDs) now exceeds 190, including 155 involving known genes, 33 with unidentified genes, and eight "multifactorial" strains. METHODS: The emerging patterns of mouse NTDs are considered in relation to the unknown genetics of the common human NTDs, anencephaly, and spina bifida aperta. RESULTS: Of the 150 mouse mutants that survive past midgestation, 20% have risk of either exencephaly and spina bifida aperta or both, parallel to the majority of human NTDs, whereas 70% have only exencephaly, 5% have only spina bifida, and 5% have craniorachischisis. The primary defect in most mouse NTDs is failure of neural fold elevation. Most null mutations (>90%) produce syndromes of multiple affected structures with high penetrance in homozygotes, whereas the "multifactorial" strains and several null-mutant heterozygotes and mutants with partial gene function (hypomorphs) have low-penetrance nonsyndromic NTDs, like the majority of human NTDs. The normal functions of the mutated genes are diverse, with clusters in pathways of actin function, apoptosis, and chromatin methylation and structure. The female excess observed in human anencephaly is found in all mouse exencephaly mutants for which gender has been studied. Maternal agents, including folate, methionine, inositol, or alternative commercial diets, have specific preventative effects in eight mutants and strains. CONCLUSIONS: If the human homologs of the mouse NTD mutants contribute to risk of common human NTDs, it seems likely to be in multifactorial combinations of hypomorphs and low-penetrance heterozygotes, as exemplified by mouse digenic mutants and the oligogenic SELH/Bc strain.     

创伤弧菌毒力相关因子的全基因组关联分析研究

【目的】创伤弧菌是致死率最高的弧菌物种,但目前尚无在全基因组层面挖掘毒力相关因子的研究。本研究以创伤弧菌分离来源(临床和环境)作为不同表型,通过与260株基因组序列进行关联分析,挖掘毒力相关因子,从而进一步了解创伤弧菌致病因素。【方法】对139株创伤弧菌分离株进行高通量测序,获取其全基因组序列;与公共数据库已公开发表的121株基因组整合,使用pyseer软件进行全基因组关联分析,对与不同分离来源显著相关的基因进行注释和解读。【结果】共发现11个基因与临床分离株显著相关,其中9个是本研究新发现的创伤弧菌潜在毒力相关因子。【结论】本研究使用群体基因组学和统计遗传学方法,在全基因组范围扫描挖掘了创伤弧菌毒力相关因子,为深入揭示该物种致病机制、设计新的疫苗和治疗靶点提供了重要依据。     

组蛋白 H3K79同型半胱氨酸修饰位点在神经管畸形中的作用

目的:探讨人类胚胎脑组织中是否存在H3K79同型半胱氨酸修饰(H3K79Hcy)及其在神经管畸形(NTDs)中的作用。方法:通过质谱检测组蛋白H3K79是否存在同型半胱氨酸修饰位点。进一步合成包含组蛋白H3K79位点的同型半胱氨酸(Hcy)修饰的肽段,并与牛血清白蛋白(BSA)偶联后免疫兔子得到抗组蛋白H3K79Hcy多克隆抗体,并对抗体进行特异性检测;采用此抗H3K79Hcy抗体比较人类高Hcy NTDs样本和正常对照样本的H3K79Hcy水平。结果:(1)人胚胎组织组蛋白H3K79位点存在同型半胱氨酸修饰;(2)高Hcy水平NTDs脑组织中H3K79Hcy修饰水平高于正常对照(P0.05)。结论:人胚胎组织存在H3K79Hcy修饰,此修饰异常可能促进神经管畸形的发生。