维甲酸诱导小鼠神经管畸形的剂量依赖性和时间特异性

目的探索不同的维甲酸（retinoic acid,RA）剂量对小鼠神经系统发育的影响以及在特异时间点作用于小鼠胚胎时对神经管闭合的影响。方法采用C57孕鼠于GD7．5分别腹部注射5mg／kg,7．5mg／kg,20mg／kgRA,于GD10．5腹部注射20mg／kgRA,观察胎鼠表型并称量胎鼠重量,胎脑重量,测量眼距和顶距（前囟到鼠喙的距离）。结果GD7．5腹部注射5mg／kgRA的胎鼠出现无眼畸形,7．5mg／kg的胎鼠出现脑膨出,20mg／kg的吸收胎率为1005,GD10．5腹部注射20mg／kgRA的胎鼠前肢短小其中,GD7．5给予7．5mg／kgRA处理后的胎鼠脑重显著低于正常组和其他实验组;GD7．5时给予7．5mg／kgRA处理组及GD10．5时20mg／kgRA处理组的胎鼠重量显著低于正常组;GD7．5时5mg／kgRA处理组的胎鼠顶距低于正常组。结论在胚胎发育的不同时期暴露于过量的RA会影响不同器官的发育,而且RA对颅面畸形的影响呈剂量依赖性。     

灵芝孢子在预防维甲酸诱导胚胎小鼠发生神经管畸形过程中对Cdk4表达的影响

目的探讨预先服用灵芝孢子在降低维甲酸诱导胚胎小鼠发生神经管畸形过程中对依赖细胞周期蛋白激酶4(cyclindependentproteinkinase4,Cdk4)表达的影响。方法于孕期小鼠E0d时给予灵芝孢子组的孕鼠胃饲灵芝孢子溶液,8g·kg-1·d-1。实验对照组的孕鼠胃饲等量溶剂。于E7.75d时,两组孕鼠一次性胃饲维甲酸,剂量为50mg/kg体重。正常对照组孕鼠胃饲等量灵芝孢子溶剂,但是在E7.75d时,仅给予维甲酸溶剂。空白对照组孕鼠常规饲养不作任何处理。在孕期E10.5d时取出4组孕鼠的胚胎,应用免疫荧光组织化学染色和Westernblot检测胚胎神经管神经上皮Cdk4表达情况。结果实验对照组孕鼠胚胎神经管神经上皮细胞Cdk4的表达明显降低,而灵芝孢子组胚胎表达Cdk4的强度明显高于实验对照组。正常对照组与空白对照组相比Cdk4的表达无统计学意义(P>0.05)。结论灵芝孢子可能是通过增强神经管神经上皮细胞表达Cdk4降低维甲酸诱导胚胎小鼠神经管畸形的发生。     

灵芝孢子在降低维甲酸诱导胚胎小鼠发生神经管畸形过程中对Cdk4表达的影响

目的 探讨灵芝孢子在降低维甲酸诱导胚胎小鼠发生神经管畸形过程中对依赖细胞周期蛋白激酶4(CDk4)表达的影响.方法 给予实验对照组和灵芝孢子组妊娠E7.75d的小鼠一次性胃饲维甲酸,造成这2组孕鼠的胚胎出现神经管畸形.然后,给予灵芝孢子组孕鼠胃饲灵芝孢子溶液.在孕期E10.5d时取出2组孕鼠的胚胎,应用免疫荧光组织化学染色和Western blotting检测胚胎神经管神经上皮依赖细胞周期蛋白激酶4(Cdk4)的表达情况.结果 在灵芝孢子组可以观察到形态正常的胚胎中,神经管的神经上皮细胞有Cdk4的表达,其表达水平与空白对照组及正常对照组相比较没有明显差异.在灵芝孢子组形态异常的胚胎中,其神经管的神经上皮细胞也有cdk4的表达,但其表达水平与空白对照组及正常对照组相比较是低的.结论 灵芝孢子可能是通过促进神经管神经上皮细胞上调Cdk4的表达来减轻维甲酸诱导的胚胎小鼠神经管畸形.     

Dishevelled2 和Vangl2表达与过量维甲酸致昆明小鼠神经管畸形的相关性

目的 探讨Dishevelled2和Vangl 2蛋白与过量维甲酸致昆明小鼠神经管畸形发生的关系。 方法 50只昆明孕小鼠随机分为对照组和实验组。孕775d时,实验组一次性胃饲30mg/kg致畸量维甲酸（RA）,对照组胃饲等量溶剂,服药后4h、18h、42h、66h及90h分别取胚胎,常规石蜡切片,采用原位杂交和免疫组织化学技术,分别检测胚胎神经管组织中Dishevelled2蛋白和Vangl2 mRNA及蛋白的表达水平变化。 结果 两种蛋白均存在于对照组及实验组不同发育阶段的小鼠神经管上皮,且两者的表达变化趋势有所不同。与对照组相比,实验组Dishevelled2蛋白在RA处理18h、42h时表达水平明显降低(P≤0.05),66h时表达升高(P≤0.05),90h时两组表达水平无明显差异;Vangl2 mRNA在灌服RA 4h、18h时表达水平明显低于对照组(P≤0.05),42h时两组表达水平无明显差异,66h时表达显著高于对照组(P≤0.05)。Vangl2蛋白在RA处理18h、42h时表达显著降低(P≤0.05),66h时两组表达水平无明显差异,90h时表达显著高于对照组(P≤0.05)。 结论 过量RA可能通过调节Dishevelled2和Vangl2表达干扰正常胚胎神经管的闭合过程。     

维甲酸受体α/β及β-连环素在维甲酸致小鼠神经管畸形中的表达变化

目的 探讨维甲酸受体α/β(RARα/β)及β-连环素(β-catenin)在维甲酸(RA)致小鼠胚胎神经管畸形发生过程中的表达变化及意义.方法 100只昆明小鼠随机分为实验组和对照组,分别通过半定量RT-PCR及Western blotting检测RARα/β及β-catenin的表达变化.结果 RARα及RARβ蛋...     

P>维甲酸受体α/β及β-连环素在维甲酸致小鼠神经管畸形中的表达变化  /P>

目的 探讨维甲酸受体α/β(RARα/β)及β-连环素(β-catenin)在维甲酸(RA)致小鼠胚胎神经管畸形发生过程中的表达变化及意义.方法 100只昆明小鼠随机分为实验组和对照组,分别通过半定量RT-PCR及Western blotting检测RARα/β及β-catenin的表达变化.结果 RARα及RARβ蛋白表达水平在RA灌服后18h、42h时明显低于正常对照组(P＜0.01);66h、90h时RARα蛋白表达水平与对照组相比无显著性差异,RARβ蛋白表达水平则显著高于正常对照组(P＜0.05).β-catenin mRNA水平在RA灌服后4h和18h时明显低于正常对照组(P＜0.05),42h时与对照组相比无显著性差异,66h时其表达水平明显高于对照组(P＜0.05);β-catenin蛋白表达水平在RA灌服后18h和42h时显著低于正常对照组(P＜0.01),66h时与对照组相比无显著性差异,90h时其表达水平显著高于正常对照组(P＜0.01).结论 RA改变了小鼠胚胎神经管中RARα/β和β-catenin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RA诱导的小鼠神经管畸形的发生.     

全反式维甲酸诱导骨骼畸形大鼠血清及羊水内碱性成纤维细胞生长因子的表达简

目的探究应用全反式维甲酸诱导骨骼畸形大鼠血清及羊水内碱性成纤维细胞生长因子（FGF2）的表达。方法选择孕10d的Wistar大鼠50只,体质量250～300g。分实验组和对照组.每组各25只。实验组予溶有全反式维甲酸的大豆油（40mg／mL）,按照135mg/hg以灌胃方式给药制作骨骼畸形胎鼠模型;对照组给予等体积的大豆油。孕20d时处死母鼠,采集母鼠血液,实验组每窝选择骨骼畸形胎鼠4只,对照组每窝随机选取胎鼠4只,抽取胎鼠羊水标本,应用酶联免疫吸附分析测定血液及羊水内FGF2浓度：利用游标卡尺测量胎鼠头臀长,双前肢各段及双后肢的长度。结果实验组胎鼠出现四肢发育不良、脊柱裂、下颌裂等畸形。实验组胎鼠头臀长、双前肢各段及双后肢长度与对照组相比.差异具有统计学意义（P〈0.05）。实验组母鼠血液及胎鼠羊水内FGF2的浓度与对照组相比[（24.124±1.271）pg／mL vs（27.451±2.026）pg／mL,（23.918±0.369）pg／mL vs（27.305±2.125）pg／mL],差异具有统计学意义（P〈0.05）。结论全反式维甲酸诱导骨骼畸形大隐.FGF2表茯情况低干骨骼发育正常的大鼠。     

全反式维甲酸诱导小鼠碱性磷酸酶基因启动子区染色体重构

背景：碱性磷酸酶基因是成骨细胞分化和骨形成的重要标志。在C3H10T1/2细胞中,全反式维甲酸可通过核受体上调小鼠碱性磷酸酶的表达,与MAPK通路无关。 目的：从染色体结构调控方面揭示全反式维甲酸上调碱性磷酸酶表达的分子机制。 方法：10^-6 mol/L全反式维甲酸处理C3H10T1/2细胞0,1,6,12 h,DNA酶Ⅰ超敏感实验确定全反式维甲酸调控区域的位置,染色质免疫共沉淀实验检验全反式维甲酸处理细胞后一系列转录相关因子与全反式维甲酸调控区域结合的量效关系以及时相分布。 结果与结论：DNA聚合酶Ⅰ超敏感实验表明,小鼠碱性磷酸酶启动子转录起始位点上游约520 bp附近为潜在的全反式维甲酸调控区域;染色质免疫共沉淀实验表明,全反式维甲酸对小鼠碱性磷酸酶的上调作用是通过一系列转录相关因子的时序性共同作用来实现。表明全反式维甲酸诱导小鼠碱性磷酸酶基因转录的过程中伴随着染色质重构和组蛋白的修饰作用。     

9-顺式维甲酸对甲状腺鳞癌细胞周期及CyclinD1、CDK4表达的影响

 摘 要：目的 探讨9-顺式维甲酸（9-cisRA）对甲状腺鳞癌细胞株SW579细胞周期及周期因子表达的影响。方法以终浓度分别为为10-7 mol/L、10-6 mol/L、5×10-6 mol/L、10-5 mol/L 的9-cisRA处理SW579细胞株,采用MTT比色法测定细胞增殖活性;流式细胞仪检测细胞周期;用RT-PCR方法检测SW579细胞CDK4、CyclinD1mRNA的表达水平;用Western blotting检测SW579细胞CDK4、CyclinD1的蛋白表达。结果 9-cisRA作用后,MTT结果显示,各组细胞均出现显著的生长抑制作用,生长抑制率明显升高,并呈剂量依赖性。流式细胞仪分析,各组细胞均随着药物浓度的升高,S期细胞在细胞周期中所占比例逐渐减少,G1期细胞则明显增加（P<0.01）,细胞滞留在G1期。RT-PCR检测结果表明,经不同浓度9-cisRA处理的细胞CDK4 mRNA表达水平没有明显变化;CyclinD1的表达水平明显下调;Western blotting检测结果表明经不同浓度9-cisRA处理的细胞CDK4蛋白表达水平没有明显变化,CyclinD1蛋白表达水平明显下降。结论9-cisRA对SW579细胞有显著的生长抑制作用,可能与抑制CyclinD1的表达,从而将细胞阻滞于G1期有关。     

PGC1α协同PPARγ调节CIDEC基因在小鼠脂肪细胞系中表达

 目的 鉴定CIDEC的表达能够被PPARγ和PGC1α共激活, 检测其在脂肪代谢中的重要调控作用。方法 构建5’删切和顺式原件突变的启动子,运用HepG2细胞中双荧光报告基因实验检测PPARγ和PGC1α对CIDEC的共激活作用并确定顺式作用原件。运用PPARγ特异激动剂pioglitazone刺激3T3-L1细胞检测CIDEC的mRNA水平。在3T3-L1细胞系中,过表达CIDEC,检测脂肪合成及能量代谢相关基因的mRNA水平。通过腺病毒感染在小鼠肝原代细胞中过表达CIDEC,检测肝脏细胞中脂肪变化。结果 PPARγ和PGC1α能够显著激活CIDEC的启动子。过表达CIDEC后在脂肪细胞中脂类合成相关基因FAS上调3.47±0.17倍（p<0.05）,ACC上调3.95±0.57倍（p<0.05）,在肝原代细胞中CIDEC促进脂滴的生成。结论 CIDEC过表达能够被PPARγ促进并且被PGC1α共激活,在体内起到促进脂肪积累的作用。     

The role of retinoic acid in the morphogenesis of the neural tube

We have examined the role of the signalling molecule, retinoic acid, in the process of neurulation and the subsequent growth and differentiation of the central nervous system using quail embryos that have developed in the absence of retinoic acid. Such retinoic acid-free embryos undergo abnormal neural tube formation in terms of its shape and structure, but the embryos do not display spina bifida or exencephaly. The neural tubes have a wider floor plate, a thicker roof plate and a different dorsoventral shape. Phalloidin staining and electron microscopy revealed alterations in the actin filaments and the junctional complexes of the cell layer lining the lumen. Initially the neural tubes proliferated at the same rate as normal, but later the proliferation rate declined drastically and neuronal differentiation was highly deficient. There were very few motoneurons extending neurites into the periphery, and within the neural tube axon trajectories were chaotic. These results reveal several functions for retinoic acid in the morphogenesis and growth of the neural tube, many of which can be explained by defective notochord signalling, but they do not suggest that this molecule plays a role in neural tube closure.     

应用银染mRNA差异显示法寻找高温致神经管畸形差异表达基因

目的 :寻找高温致神经管畸形的差异表达基因。方法 :在高温致神经管畸形的动物模型上 ,分别于高温处理后 2 4、48和 72小时 ,提取鼠胚神经管组织总RNA和正常对照组鼠胚相应时间的神经管组织总RNA ,反转录合成cDNA第一链后进行差异显示PCR扩增 ,采用PAGE和银染技术显示差异条带 ,回收差异条带并经PCR二次扩增后 ,用点杂交方法筛除假阳性条带 ,再用Northern印迹杂交进一步鉴定。结果 :在高温致畸的鼠胚神经管组织中筛选到一个特别明显的差示cDNA片段N3 2 ,该片段所在基因在高温致畸的胚胎神经管组织中的表达远低于正常同龄胚的神经管组织。结论 :N3 2片段所在基因的低表达与高温致神经管畸形相关。     

Novel mutations in VANGL1 in neural tube defects

Neural tube defects (NTDs) are severe congenital malformations caused by failure of the neural tube to close during neurulation. Their etiology is complex involving both environmental and genetic factors. We have recently reported three mutations in the planar cell polarity gene VANGL1 associated with NTDs. The aim of the present study was to define the role of VANGL1 genetic variants in the development of NTDs in a large cohort of various ethnic origins. We identified five novel missense variants in VANGL1, p.Ser83Leu, p.Phe153Ser, p.Arg181Gln, p.Leu202Phe and p.Ala404Ser, occurring in sporadic and familial cases of spinal dysraphisms. All five variants affect evolutionary conserved residues and are absent from all controls analyzed. This study provides further evidence supporting the role of VANGL1 as a risk factor in the development of spinal NTDs. © 2009 Wiley‐Liss, Inc.     

神经管畸形患儿还原叶酸载体基因和叶酸之间交互作用

目的 探讨神经管畸形(neural tube defects,NTDs)患儿还原叶酸载体基因(reduced folate carrier gene,RFCI）A80G多态性与母亲孕早期未增补叶酸之间的关联性,为寻找NTDs危险因素的遗传易感标志物提供流行病学依据。方法采用限制性片段长度多态性-聚合酶链反应方法,对104个NTDs患儿及其母亲和100名正常儿童及其母亲的外周血DNA进行RFCI第80位单核苷酸多态性检测,通过病例对照研究,调查了后代RFCI A80G基因型与母亲孕期前后增补叶酸之间的基因环境交互作用。结果 RFCI GG基因型的子代发生NTDs危险高于AA基因型子代(OR=2．56,95％CI=1．04～6．36);母亲孕早期不增补叶酸,生育NTDs的危险高于增补叶酸的母亲(OR=7．69,95％CI=2．86～21．75);母亲孕期未增补叶酸,其子代GG基因型,发生NTDs的危险是AA基因型的3．30倍(95％CI=1．15～9．65);在叶酸和RFCI基因交互作用研究中,母亲未增补叶酸和子代GG基因型同时存在,发生NTDs的危险是8．80(95％CI=2．86～29．82),交互作用系数为1．45,,结论 在中国人群中,RFCI GG基因型可能是NTDs发生的遗传易感基因之一,子代RFCI GG基因型与母亲孕期叶酸缺乏之间存在交互作用,可能增加NTDs的发病危险。     

Exploring gene—gene interactions in the etiology of neural tube defects

The role of susceptibility genes in the etiology of birth defects is unclear, but may involve in some cases multiple alleles at multiple loci. We suggest a simple epidemiologic approach to explore gene-gene interactions, and use it to reevaluate data from a recent case-control study on the possible association of neural tube defects (NTDs) with specific mutations of two genes, 5,10-methylene-tetrahydrofolate reductase (MTHFR) and cystathionine-β synthase (CBS). We found that, compared with the common genotype, homozygosity for the MTHFR mutation alone was associated with a two-fold increased risk for NTDs, while homozygosity for the CBS mutation alone was not a risk factor. However, homozygous individuals for the mutations at both loci had a five-fold greater risk for NTDs than those with the reference genotype. Though the original study was too small to detect statistically significant differences among most of the risk estimates, these results, if confirmed by independent and larger studies, suggest that gene-gene interaction may play a role in modulating the susceptibility to NTDs in a proportion of affected individuals. This approach, moreover, could be a valuable adjunct to the study of gene-gene interactions in the etiology of human disease.     

Contribution of VANGL2 mutations to isolated neural tube defects

Kibar Z, Salem S, Bosoi CM, Pauwels E, De Marco P, Merello E, Bassuk AG, Capra V, Gros P. Contribution of VANGL2 mutations to isolated neural tube defects. Vangl2 was identified as the gene defective in the Looptail (Lp) mouse model for neural tube defects (NTDs). This gene forms part of the planar cell polarity (PCP) pathway, also called the non‐canonical Frizzled/Dishevelled pathway, which mediates the morphogenetic process of convergent extension essential for proper gastrulation and neural tube formation in vertebrates. Genetic defects in PCP signaling have strongly been associated with NTDs in mouse models. To assess the role of VANGL2 in the complex etiology of NTDs in humans, we resequenced this gene in a large multi‐ethnic cohort of 673 familial and sporadic NTD patients, including 453 open spina bifida and 202 closed spinal NTD cases. Six novel rare missense mutations were identified in seven patients, five of which were affected with closed spinal NTDs. This suggests that VANGL2 mutations may predispose to NTDs in approximately 2.5% of closed spinal NTDs (5 in 202), at a frequency that is significantly different from that of 0.4% (2 in 453) detected in open spina bifida patients (p = 0.027). Our findings strongly implicate VANGL2 in the genetic causation of spinal NTDs in a subset of patients and provide additional evidence for a pathogenic role of PCP signaling in these malformations.     

CD1+ cells in mothers of stillborn infants with neural tube defects

In healthy individuals, CD1+ cells are found in thymic tissue, but not in peripheral blood. The thymus, as a key organ of the neuroendocrine system, frequently shows gross abnormalities in infants with neural tube defects. In order to study the immuno-logic significance of fetal thymic findings, maternal T-lymphocyte subpopulations were investigated. In 10 mothers of healthy new-borns, 5 mothers of stillborn infants who had no gross abnormalities, and 5 mothers of stillborn infants with neural tube defects, CD1+, CD3+, CD4+, and CD8+ cells were studied. Only the mothers of the infants with open neural tube defects showed CD1 + cells in their peripheral blood. © 1995 Wiley-Liss, Inc.