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.     

Nestin在高温致畸神经管的表达及多种维生素和微量元素对神经管畸形的保护作用

本研究旨在探讨高温致神经管畸形(NTD)中巢蛋白(nestin)的表达规律及多种维生素和微量元素对NTD的保护作用。孕鼠随机分为实验组和对照组。实验组又进一步分为3组,A组:孕前第8d开始灌服多种维生素和微量元素;B组:孕后第1d开始灌服;C组:不灌服。孕鼠分别于孕第8d高温处理,对照组不灌服多种维生素和微量元素,也不进行高温处理。对以上各组胎鼠进行形态学观察,计算神经管畸形发生率;利用免疫荧光染色观察nestin在胚胎不同发育时期的神经上皮细胞中的表达规律。结果显示,在实验组中,C组神经管畸形发生率最高,为77.5%;各组鼠胚神经上皮细胞胞质中nestin呈阳性染色,实验C组的染色明显弱于其它组;不同发育阶段各组染色强度不同。结果提示,nestin低表达是神经管畸形发生的重要因素,多种维生素和微量元素对高温致神经管畸形有保护作用。     

神经细胞黏附分子1和骨调蛋白在神经管畸形中的变化

目的初步探究神经细胞黏附分子1和骨调蛋白在神经管畸形中的变化。方法20只健康成年SD孕大鼠随机分为正常对照组和神经管畸形实验组,每组10只。通过胃饲维甲酸建立胚胎大鼠神经管畸形模型,检查了神经管畸形鼠体重的变化;用HE染色观察神经管畸形鼠神经管结构的变化;通过Western blot分析神经管畸形鼠组织中神经细胞黏附分子1和骨调蛋白的变化。结果神经管畸形鼠形态偏小,体质量为(0.34±0.45)g,明显低于正常对照组(0.53±056)g。胚胎鼠在第9天神经管处于未闭合状态,在第11天已完全闭合。胚胎第9天和11天神经管畸形大鼠的神经细胞黏附分子1比正常组表达均明显增加(P0.01)。骨调蛋白在胚胎第9 d神经管畸形大鼠中的表达比正常明显增高(P0.01),但在胚胎第11天的表达与正常相比没有差异(P0.05)。结论神经细胞黏附分子1和骨调蛋白的变化可能与神经管畸形形成有密切关系。     

维甲酸诱导的小鼠神经管畸形模型中神经系统相关基因的表达情况分析

 摘要: 目的: 研究在全反式维甲酸诱导小鼠神经管畸形模型中神经系统相关基因（ASH2L,HDAC4,NSPC1与DOK5）的表达变化。方法: 取8只E8.5的C57/BL6孕鼠随机分为两组：对照组（4只），模型组（4只）。对照组腹腔注射橄榄油，模型组腹腔注射全反式维甲酸；E13.5取胚胎。采用Real-time PCR检测两组小鼠脑和脊髓中ASH2L,HDAC4,NSPC1与DOK5的mRNA表达；采用Western Blotting技术检测两组小鼠脑和脊髓中ASH2L,HDAC4,NSPC1与DOK5的蛋白表达情况。结果：全反式维甲酸可诱导小鼠出现典型神经管畸形；与对照组相比，全反式维甲酸处理组的致畸胚胎脑和脊髓中,ASH2L HDAC4, NSPC1和DOK5的mRNA表达有所下降（P<0.05）；同时这些基因的蛋白水平也显著下降 (P<0.05)。结论：ASH2L,HDAC4,NSPC1和DOK5可能是抑制全反式维甲酸致神经管畸形的潜在基因。     

尼古丁影响鼠胚神经管Nestin和S-100β表达

目的 探讨生殖致畸期宫内暴露尼古丁是否影响鼠胚神经管nestin和S—100g表达。方法 建立妊娠期宫内暴露尼古丁的大鼠模型,免疫组织化学法观察孕13d神经管顶板基板及16d鼠胚中缝组织分化程度的改变。结果 尼古丁组孕13d大鼠胚胎菱脑顶板未完全闭合,部分区域无神经上皮覆盖,nestin阳性神经上皮细胞数量明显比正常对照组少（P〈0．05）,排列紊乱;孕16d鼠胚中缝处尼古丁组S-100β表达较对照组为弱（P〈0．05）;神经上皮细胞排列不规则,核仁不清晰,胞质内可见空泡,细胞间隙增宽。结论生殖致畸期内暴露尼古丁影响神经管菱脑区顶板、基板神经组织正常发育,延迟神经管闭合;延迟孕16d鼠胚中缝处神经组织的完全闭合。     

维甲酸对胎鼠Vangl1及Vangl2基因表达的影响

神经管畸形的发生是由遗传因素与环境因素共同作用产生的。平面细胞极性途径（planar cell polarity，PCP）对脊椎动物神经胚形成非常重要。该途径基因的任何突变都可能导致神经管不能闭合，与人类的颅脊柱裂相似。但是，环境因素是否影响、如何影响PCP途径基因的表达到目前仍不清楚。目的研究维甲酸对胎鼠Vangl1及Vangl2基因表达的时空特征的影响。方法全反式维甲酸橄榄油混悬液按120mg／kg在BALB／C小鼠怀孕第9．5天（E9．5）（B组）或E10．5（C组）灌胃，A组仅用橄榄油灌胃。取E9．5、E10．5、E11．5、E13．5、E15．5、E17．5、E19．5胎鼠，Vangl1及Vangl2基因表达量采用逆转录酶PCR（RT—PCR）检测，其时空表达采用全胚胎原位杂交（WISH）检测。结果结果显示，正常情况下Vangl1及Vangl2基因在整个神经胚形成过程中都有强表达。而B组胎鼠从脑到后神经孔Vangl1及Vangl2基因表达显著下调。C组较复杂，二基因表达在全胚胎（从E11．5到E13．5）及神经管脊柱区（从E15．5到E19．5）显著下调并维持低表达，但在神经管的颅区（E15．5到E19．5）仅中度下调。结论：结果提示，维甲酸诱导的神经管畸形的发生与Vangl1及Vangl2基因转录子下调有关。     

HMGB1在高温致金黄地鼠神经管畸形神经上皮细胞中的表达变化

为了探讨高温致神经管畸形(NTDs)作用的分子机制,为预防人类NTDs的发生提供理论依据,本研究在高温致金黄地鼠NTDs模型的基础上,研究HMGB1在高温致金黄地鼠NTDs神经上皮细胞中的表达变化。将孕鼠随机分为实验组和对照组,分别于水浴处理后8、16、24、40、64 h(相当于胚龄第8、8.5、9、9.5、10.5 d)处死孕鼠,剖腹取鼠胚,制备石蜡切片,应用免疫荧光染色技术检测NTDs发生过程中HMGB1在神经上皮细胞中的表达变化。结果显示:对照组和模型组孕鼠在水浴处理后8、16、24h,HMGB1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40、64 h,HMGB1表达部位出现了由胞浆向胞核的转移;高温处理后,HMGB1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。上述结果提示,HMGB1的表达变化与神经管发育密切相关,其表达降低是高温致神经管畸形发生的重要途径之一。     

大鼠胚胎神经干细胞黑质内移植后的存活及分化

目的：观察胚胎神经于细胞脑内移植后的存活及生长分化状况。方法：从孕11d(E11d)大鼠胚胎神经管获取神经上皮细胞，经神经巢蛋白(nestin)染色鉴定干细胞；同时植入同种大鼠黑质内。于移植后7d、14d取脑，用神经元特异烯醇化酶(NSE)、酪氨酸羟化酶(TH)免疫组化方法检测移植细胞的存活及分化状况。结果：E11d神经管上皮细胞多数呈nestin染色阳性，黑质内移植后增殖形成细胞团并随时间延长而增大。免疫组化染色显示移植细胞团内有NSE及TH免疫阳性细胞。结论：胚胎神经上皮细胞多数为神经干细胞，黑质内移植后可以存活并分化为多巴胺能神经元。     

脑源性神经营养因子在体外诱导SH-SY5Y细胞分化过程中的作用

目的 探讨不同浓度的脑源性神经营养因子(BDNF)在体外诱导SH-SY5Y细胞分化时,对G2期及其前后细胞周期相关蛋白mRNA表达的影响.方法 采用全反式维甲酸(RA)和低(1μg/L)、中(10μg/L)、高(100μg/L)3种不同浓度BDNF在无血清培养液中相继诱导SH-SY5Y细胞分化,形成均一的具有神经元形态的细胞.以一步法实时定量RT-PCR检测其G2期细胞周期相关蛋白mRNA的表达,荧光激活细胞分类术(FACS)检测各组细胞时相.结果 在各浓度BDNF组中周期蛋白B1(cyclin B1)的mRNA含量与对照组及RA组比较均明显降低(P<0.05);仅高浓度组cyclin B2和周期蛋白依赖性激酶1(Cdk1)的mRNA含量显著降低(P<0.05),;低浓度和中浓度BDNF组Cdk5 mRNA含量均高于RA组(P<0.05).BDNF各浓度组处于G1期的细胞百分率均显著高于RA和对照组(P<0.01),而处于S期的细胞百分率均显著低于对照组(P<0.01),且低浓度及高浓度组同时低于对照组(P<0.01).结论 提示BDNF在有效促进SH-SY5Y细胞进一步分化的同时,没有引起G2期及其前后细胞周期相关蛋白mRNA表达的异常升高,BDNF无诱导细胞凋亡的危险,可能有减缓AD进程的作用,并且呈剂量依赖性.     

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。     

Specific isoforms of protein kinase C are essential for prevention of folate-resistant neural tube defects by inositol

A proportion of neural tube defects (NTDs) can be prevented by maternal folic acid supplementation, although some cases are unresponsive. The curly tail mutant mouse provides a model of folate-resistant NTDs, in which defects can be prevented by inositol therapy in early pregnancy. Hence, inositol represents a possible novel adjunct therapy to prevent human NTDs. The present study investigated the molecular mechanism by which inositol prevents mouse NTDs. Activation of protein kinase C (PKC) is known to be essential, and we examined neurulation-stage embryos for PKC expression and applied PKC inhibitors to curly tail embryos developing in culture. Although all known PKC isoforms were detected in the closing neural tube, use of chemical PKC inhibitors identified a particular requirement for 'conventional' PKC isoforms. Peptide inhibitors offer selective inhibition of individual PKCs, and we demonstrated isoform-specific inhibition of PKC in embryonic cell cultures. Application of peptide inhibitors to neurulation-stage embryos revealed an absolute dependence on the activity of PKCbetaI and gamma for prevention of NTDs by inositol, and partial dependence on PKCzeta, whereas other PKCs (alpha, betaII delta, and epsilon) were dispensable. To investigate the cellular action of inositol and PKCs in NTD prevention, we examined cell proliferation in curly tail embryos. Defective proliferation of hindgut cells is a key component of the pathogenic sequence leading to NTDs in curly tail. Hindgut cell proliferation was stimulated specifically by inositol, an effect that required activation of PKCbetaI. Our findings reveal an essential role of specific PKC isoforms in mediating the prevention of mouse NTDs by inositol.     

Gene-environment interactions in the causation of neural tube defects: folate deficiency increases susceptibility conferred by loss of Pax3 function

Risk of neural tube defects (NTDs) is determined by genetic and environmental factors, among which folate status appears to play a key role. However, the precise nature of the link between low folate status and NTDs is poorly understood, and it remains unclear how folic acid prevents NTDs. We investigated the effect of folate level on risk of NTDs in splotch (Sp(2)(H)) mice, which carry a mutation in Pax3. Dietary folate restriction results in reduced maternal blood folate, elevated plasma homocysteine and reduced embryonic folate content. Folate deficiency does not cause NTDs in wild-type mice, but causes a significant increase in cranial NTDs among Sp(2)(H) embryos, demonstrating a gene-environment interaction. Control treatments, in which intermediate levels of folate are supplied, suggest that NTD risk is related to embryonic folate concentration, not maternal blood folate concentration. Notably, the effect of folate deficiency appears more deleterious in female embryos than males, since defects are not prevented by exogenous folic acid. Folate-deficient embryos exhibit developmental delay and growth retardation. However, folate content normalized to protein content is appropriate for developmental stage, suggesting that folate availability places a tight limit on growth and development. Folate-deficient embryos also exhibit a reduced ratio of s-adenosylmethionine (SAM) to s-adenosylhomocysteine (SAH). This could indicate inhibition of the methylation cycle, but we did not detect any diminution in global DNA methylation, in contrast to embryos in which the methylation cycle was specifically inhibited. Hence, folate deficiency increases the risk of NTDs in genetically predisposed splotch embryos, probably via embryonic growth retardation.     

用原位缺日翻译法比较两种细胞死亡的DNA状态

用原位缺日翻译法比较两种细胞死亡的ＤＮＡ状态（北京中医药大学组织学胚胎学教研室．北京１０００２９牛建昭，小路武彦，中根一穗）ＤＮＡ断裂存在于不同细胞周期中（增殖、分化、死亡）。为研究不同组织细胞核内ＤＮＡ链切断（ＤＳＢ），我们采用了缺口翻译法（ＩＮＴ...     

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.     

Valproic acid induced abnormal development of the central nervous system of three species of amphibians: implications for neural tube defects and alternative experimental systems.

Embryos of Ambystoma mexicanum, Xenopus laevis, and Hyperolius viridiflavus taeniatus were exposed to various concentrations of valproic acid (VPA: 0.1, 1.5, 10 mM) from blastula stage (S) 9 on up to advanced gastrulation of control embryos (S 11 1/2-12). At 10 and 5 mM VPA early development was affected in all species tested. However, the most pronounced effects occurred in Ambystoma: the neural folds appeared delayed and showed a flattened and wavy shape; the neural tube was not formed and embryos successively died. In Xenopus and Hyperolius (10, 5 mM VPA) the beginning of gastrulation was delayed up to neurulation of control embryos. In Xenopus many of the embryos completed neurulation, whereas some embryos exposed to 10 mM VPA showed neural tube defects (NTDs) of different type and degree (open neural tube at different regions of the dorsum). In Hyperolius neural folds arose around the blastoporus and fused later on (earlier in embryos treated with 5 mM VPA), but the shape of these embryos was abnormal and the development was not continued (pronounced effect at 10 mM VPA). Comparing the three species, Xenopus proved to be the least sensitive species (at 5 mM VPA 14.2% NTDs of total malformations compared to 100% in the other species). The most sensitive species, Ambystoma, developed head-oedema at 1 mM VPA, whereas the anurans were not affected. Our results suggest a similar mechanism of VPA-induced NTDs in mammals and amphibians.