磷酸化的JNK和p38在高温致神经管畸形上皮细胞中的表达变化

目的 探讨高温致神经管畸形（neural tube defects,NTD）的分子机制,为防治神经管畸形提供理论依据.方法 利用高温致金黄地鼠NTD动物模型,应用免疫荧光染色技术,观察NTD发生过程中磷酸化c-Jun氨基末端活化蛋白激酶（p-JNK）和磷酸化p38（p-p38）在胚胎神经管上皮的表达变化.结果 p-JNK和p-p38的免疫阳性产物表达于胚胎神经管上皮细胞及其周围间充质细胞的胞浆中,高温后不同时间点胚胎神经管上皮细胞内的p-JNK和p-p38的表达量均较对照组减弱.结论 磷酸化JNK和p38参与了神经管的正常发育,其表达量降低可能是高温致NTD发生的重要途径之一.     

热休克蛋白在高温致神经管畸形中的表达

目的：检测热休克蛋白在高温致神经管畸形中的表达状况,以探讨高温致神经管畸形的机制。方法：在高温致金黄地鼠神经管畸形的动物模型上,利用免疫组织化学(SABC法)方法,检测高温致神经管畸形中,热休克蛋白(HSP70和HSP90)在神经上皮细胞及周围间充质细胞中的表达状况;同时利用地高辛标记的寡核苷酸探针进行原位杂交,检测HSP70 mRNA和HSP90 mRNA在神经上皮细胞及周围间充质细胞中的转录状况。结果：高温处理后2h,鼠胚神经上皮细胞及周围间充质细胞HSP70、HSP90的表达与正常对照组相比明显增强,8h和16h的表达达到高峰,24h后与对照组水平一致。原位杂交结果显示,高温处理后2h神经上皮细胞及周围间充质细胞中出现HSP70 mRNA及HSP90mRNA杂交阳性信号,8h阳性信号最强,16h后阳性信号减弱,至24h后未见阳性信号。结论：高温可引起神经上皮细胞及其周围间充质细胞HSP70和HSP90应激性表达,这可能是胚胎受到高温作用后发生的一种保护性反应。     

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.     

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.     

大鼠体内视网膜神经节细胞诱向因子(RGNTF)及其受体的免疫组织化学定位

本文用ＲＧＮＴＦ单克隆抗体及抗独特型单克隆抗体的免疫组织化学反应,对ＲＧＮＴＦ及其受体在 大鼠体内的分布进行了研究．结果显示,大鼠的肾脏、肾上腺、下颌下腺、胃底腺,以及睾丸的生精细胞对 ＲＧＮＴＩＦ均呈现强阳性免疫反应,并对ＲＧＮＴＦ抗独特型单克隆抗体也呈现阳性免疫反应,表明 ＲＧＮＴＦ及其受体有较广泛的分布,这种情况与神经生长因子（ＮＧＦ）及睫状节神经诱向（营养）因子 （ＣＮＴＦ）相类似．但是,ＲＧＮＴＦ及其受体的分布特点和ＮＧＦ、ＣＮＴＦ的分布是不完全相同的,提示作者 分离的ＲＧＮＴＦ与ＮＧＦ和ＣＮＴＦ不是同源物。这样肾上腺皮质、下颌下腺的浆液腺泡及导管上皮细胞、 胃底腺上皮细胞和生精细胞不仅能够产生ＲＧＮＴＦ,也能合成ＲＧＮＴＦ受体。因此,它们对ＲＧＮＴＦ可能 有自分泌的功能,ＲＧＮＴＦ对这些细胞可能有自身调节的效应。     

大鼠体内视网膜神经节细胞透向因子（RGNTF）及其受体的免疫组织化学定位

本文用ＲＧＮＴＦ单克隆抗体及抗独特型单克隆抗体的免疫组织化学反应,对ＲＧＮＴＦ及其受体在大鼠体仙的分布进行了研究。结果显示,大鼠的肾脏、肾上腺、下颌下腺、胃底腺,以及睾丸丸的生精细胞对ＲＧＮＴＦ均呈现强阳性免疫反应,并对ＲＧＮＴＦ抗独特型单克隆抗体也呈现阳性免疫反应,表明ＲＧＮＴＦ及其受体有较广泛的分布,这种情况与神经生长因子（ＮＧＦ）及睫状节神经诱向（营养）因子（ＣＮＴＦ）相类似。但是,ＲＧＮＴ     

N42－A对神经生长因子诱导PC12细胞分化的抑制作用

研究表明,缺乏神经生长因子（ＮＧＦ）的营养支持是Ａｌｚｈｅｉｍｅｒ＇ｓ等神经元退行性疾病发生发展的重要原因,而ＮＧＦ和／或ＮＧＦ受体的过度表达则与一些神经系统肿瘤的发生发展有着十分密切的因果关系。采用（１２５）Ⅰ－ＮＧＦ受体特异结合实验作为ＮＧＦ受体活性物质筛选实验模型从中药牛膝中筛选出了能强烈地抑制（１２５）Ⅰ－ＮＧＦ受体结合的活性成分Ｎ４２－Ａ（ⅠＣ（５０）＝６．１８±３．４３,ｎ＝４）;细胞培养实验表明,Ｎ４２－Ａ对ＮＧＦ诱导大鼠嗜铬神经瘤ＰＣｌ２细胞的分化也具有很强的剂量依赖性抑制作用（对０．１ｎｍｏｌ／Ｌ和０．２ｎｍｏｌ／ＬＮＧＦ诱导的大鼠嗜铬神经病ＰＣ１２细胞轴突生长的半数抑制浓度分别为６μｇ／ｍＬ和２１μｇ／ｍＬ）。这表明,Ｎ４２－Ａ是神经元上介导ＮＧＦ诱导轴突生长的特异受体抑制剂,不仅对ＮＧＦ及其受体过度表达所致的神经系统肿瘤的防治具有潜在的应用价值,而且对Ａｌｚｈｅｉｍｅｒ＇ｓ等神经元退行性疾病防治药物的开发研究具有十分重要的意义。     

N42—A神经生长因子诱导PC12细胞分化的抑制作用

研究表明,缺乏神经生长因子（ＮＧＦ）的营养支持是Ａｌｚｈｅｉｍｅｒ′ｓ等神经元退行性疾病发生发展的重要原因,而ＮＧＦ和／或ＮＧＦ受体的过度表达则与一些神经系统肿瘤的发生发展有着十分密切的因果关系。采用＾１２５Ｉ－ＮＧＦ受体特异结合实验作为ＮＧＦ受体活性物质筛选实验模型从中药牛膝中筛选出了能强烈地抑制＾１２５Ｉ－ＮＧＦ受体结合的活性成分Ｎ４２－Ａ（ＩＣ５０＝６．１８±３．４３,ｎ＝４）;细胞培养实验     

人γ干扰素（IFN－γ）－表皮生长因子（EGF）融合蛋白的抗肿瘤细胞增殖作用与靶细胞的EGF受体有关

采用表皮生长因子（ＥＧＦ）受体丰富的Ａ４３１细胞进一步研究了人ＩＦＮ－γ－ＥＧＦ３融合蛋白￣〔１〕的抗细胞分裂活性,以及它和靶细胞ＥＧＦ受体的关系。结果表明,ＩＦＮ－γ－ＥＧＦ３融合蛋白的抗肿瘤细胞增殖作用明显高于其母体分子。￣１２５Ｉ－ＥＧＦ受体竞争抑制试验表明,ＩＦＮ－γ可与ＥＧＦ竞争Ａ４３１细胞的ＥＧＦ受体,而ＩＮＦ－γ－ＥＧＦ３融合蛋白的ＥＧＦ受体竞争抑制作用更为明显,说明人ＩＦＮ－γ和ＩＦＮ－γ－ＥＧＦ３融合蛋白的抗肿瘤细胞分裂活性,与靶细胞ＥＧＦ受体被竟争抑制密切相关。     

胶质细胞源性神经营养因子GDNF和Neurturin的新受体: TrnR2

胶质细胞源性神经营养因子ＧＤＮＦ和Ｎｅｕｒｔｕｒｉｎ的新受体：ＴｒｎＲ２最近,Ｎｅｕｒｔｕｒｉｎ（ＮＴＮ）———一种与胶质细胞源性神经营养因子（ＧＤＮＦ）相关的神经营养因子被发现,于是产生了一个由ＧＤＮＦ和ＮＴＮ所组成的转化生长因子（ＴＧＦ）β相关...     

bFGF、NGF、EGF及其受体在人胚神经管早期发育中的表达

研究bFGF、NGF、EGF及其受体在人胚神经管早期发育中的表达。方法 应用免疫组织化学方法和图像分析。结果显示bFGF和NGF的表达时序不同,bFGF阳性细胞出现较早,在所检测在各个发育阶段均呈阳性表达,而NGF出现较晚,随着胚龄增加,免疫阳性着色逐渐增强,bFGF分布较NGF广泛,而EGF在所检测的各个发育阶段均呈阴性。flg、TrkA、EGFR表达时序和分布相似,三者在所检测的各个发育阶段均阳性。结果表明NGF和bFGF均通过其特异性受体介导,在胚胎神经管形成和分化的不同阶段发挥着重作用,EGF及其受体的作用有待进一步研究。     

电针对局灶性脑缺血大鼠脑内神经生长因子受体trkA的影响

神经生长因子对神经元凋亡有拮抗作用,其作用方式是通过特异性受体——ｔｒｋＡ实现。为了探讨针刺对内源性抗凋亡因素的调节作用。本研究用大鼠局灶性脑缺血再灌注模型,应用免疫组织化学方法观察缺血再灌注时ｔｒｋＡ的变化及针刺对ｔｒｋＡ的影响。结果发现：缺血组对照侧大脑皮层偶见散在ｔｒｋＡ免疫阳性神经元,缺血侧大脑皮层ｔｒｋＡ阳性神经元与对照侧相比,显微镜下见无显著性差异。电针组缺血侧大脑皮层见大量ｔｒｋＡ免疫反应阳性神经元,主要分布于半影区,与对照侧及未电针的缺血侧相比,显微镜下见阳性神经元数目有差异。结果提示：电针可以诱导脑缺血时神经营养因子受体表达,调动机体内抗凋亡因素的作用。     

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。     

In vitro analysis of sympathetic neuron differentiation from chick neural crest cells

Sympathetic neuron differentiation was studied using a fluorescence histochemical assay to detect the appearance of cell-bound catecholamines. Results from in vitro organ cultures indicate that chick neural crest cells must interact with both ventral neural tube (defined throughout as the ventral neural tube plus the notochord) and somitic mesenchyme in order to differentiate into sympathoblasts. Somite, ventral neural tube, and crest were cultured transfilter in various combinations to define these tissue interactions more precisely. Results from these experiments indicate that neural crest cells must be contiguous to somite in order to differentiate into sympathoblasts, but ventral neural tube may act across a Millipore filter membrane (type TH, 25 μm thick) either on somite, crest, or both. To distinguish among these possibilities, somite was cultured transfilter to ventral tube for a short period, after which ventral tube was removed and fresh crest was added to the somite. The results from this and other experiments support the hypothesis that the ventral tube does not act directly on crest cells, but elicits a developmental change in somitic mesenchyme, which then promotes sympathoblast differentiation. To study the relationship of nerve growth factor (NGF) to the differentiation of sympathetic neurons, cultures of somite + crest were temporarily exposed transfilter to ventral tube, in the presence or the absence of exogenous NGF. The results of these and other experiments are consistent with the hypothesis that the continued presence of ventral tube is required to ensure the survival of the differentiating sympathetic neurons. With respect to this second function, ventral tube can be replaced by exogenous NGF.     

Raltitrexed’s effect on the development of neural tube defects in mice is associated with DNA damage,apoptosis, and proliferation

The causal metabolic pathway and the underlying mechanism between folate deficiency and neural tube defects (NTDs) remain obscure. Thymidylate (dTMP) is catalyzed by thymidylate synthase (TS) using the folate-derived one-carbon unit as the sole methyl donor. This study aims to examine the role of dTMP biosynthesis in the development of neural tube in mice by inhibition of TS via a specific inhibitor, raltitrexed (RTX). Pregnant mice were intraperitoneally injected with various doses of RTX on gestational day 7.5, and embryos were examined for the presence of NTDs on gestational day 11.5. TS activity and changes of dUMP and dTMP levels were measured following RTX treatment at the optimal dose. DNA damage was determined by detection of phosphorylated replication protein A2 (RPA2) and γ-H₂AX in embryos with NTDs induced by RTX. Besides, apoptosis and proliferation were also analyzed in RTX-treated embryos with NTDs. We found that NTDs were highly occurred by the treatment of RTX at the optimal dose of 11.5 mg/kg b/w. RTX treatment significantly inhibited TS activity. Meanwhile, dTMP was decreased associated with the accumulation of dUMP in RTX-treated embryos. Phosphorylated RPA2 and γ-H₂AX were significantly increased in RTX-treated embryos with NTDs compared to control. More apoptosis and decreased proliferation were also found in embryos with NTDs induced by RTX. These results indicate that impairment of dTMP biosynthesis caused by RTX led to the development of NTDs in mice. DNA damage and imbalance between apoptosis and proliferation may be potential mechanisms.     

环磷酰胺对大鼠神经上皮细胞向神经元方向分化的影响

目的研究环磷酰胺对大鼠神经上皮细胞向神经元方向分化的影响,进一步探讨其致畸作用的分子机制,为预防人类NTDs的发生提供理论依据。方法将孕鼠随机分实验组和对照组,实验组在妊娠第13天上午8-9时腹腔注射环磷酰胺(15mg/kg体重),对照组注射等量生理盐水。分别于给药后4、8、12、24、48h处死孕鼠,剖腹取出胎鼠,于10%中性福尔马林中固定,常规石蜡包埋、连续切片。应用免疫组织化学和免疫荧光技术检测β-tubulin和NeuN在NTDs发生中的表达变化。结果(1)β-tubulin免疫荧光染色结果:给药后4、8h,实验组β-tubulin阳性率与对照组相比,差异无统计学意义;给药后12、24、48h,β-tubulin阳性细胞与对照组相比减少,差异均有统计学意义。(2)NeuN免疫组织化学结果:给药后4、8、12h,实验组和对照组均未检测到NeuN阳性细胞;给药后24、48h,实验组与对照组相比,NeuN阳性细胞减少,差异均有统计学意义。结论环磷酰胺可使β-tubulin、NeuN的表达降低,干扰神经上皮细胞向神经元方向分化。     

Immunohistochemical profile of CD markers in experimental neural tube defect

ABSTRACT

Neural tube defects (NTDs) are the second most common birth defects worldwide. Stem cells play a critical role in the mechanisms underlying NTDs. We established an experimental NTD model in rats using retinoic acid (RA). We used mesenchymal and hemopoietic stem cell markers to determine their distribution in the mesenchyme in and around the neuroepithelium during the embryonic and fetal periods in both cranial and caudal regions. Adult female rats were given RA on days 5 and 10 of gestation and olive oil was administered to the control group. On days 10.5 and 15.5, embryos in the experimental and control groups were removed from the uterus. Embryos were embedded in paraffin and serial sections of the cranial and caudal neural tube were examined. We found severe cranial and caudal defects including axial rotation in the experimental groups using histochemistry. We used CD44, CD56, CD73, CD90, CD105, CD271 antibodies as mesenchymal stem cell markers and CD14, CD45 as hemopoietic stem cell markers. More CD44, CD56, CD90, CD105 and CD14 were detected during the embryonic period than the fetal period. CD73 was more frequent during the fetal period, whereas CD271 and CD45 were not significantly different. When CD44, CD56, CD73, CD90, CD105, CD271 immunostaining was found, NTDs were decreased early and increased later. We found no significant difference between CD14 and CD45. Formation of NTDs was due to deterioration of the of the neuroepithelial and surrounding stem cells. One reason for the formation of NTDs is that stem cells may develop defective cell-cell or cell-matrix interactions.