细胞外信号调节激酶系统(ERKs)在正常发育和单眼剥夺大鼠视皮层蛋白表达的研究

为了研究正常发育和单眼视觉剥夺大鼠视皮层 ERK1/ ERK2基因蛋白质表达的变化 ,本研究建立了 SD大鼠单眼视觉剥夺模型 ,以多克隆抗 ERK1/ ERK2抗体和单克隆抗双磷酸化 ERK抗体检测出生后第 1、14、2 1、2 8、45 d和成年 (90 d)正常发育和单眼视觉剥夺 (14～ 45 d)视皮层 ERK1/ ERK2蛋白表达和活性改变。结果表明 ,出生后大鼠视皮层 ERK1/ ERK2蛋白表达逐渐增加 ,至 45 d达到高峰 ,此后下降至成年达稳定水平。磷酸化 ERK蛋白表达仅见于成年大鼠视皮层。单眼视觉剥夺导致ERK1/ ERK2蛋白表达减少。提示 ERK的蛋白表达依赖于正常的视觉输入信号的刺激 ,异常的视觉经验造成表达的明显下调 ,阻断正常的发育进程。说明 ERK1和 ERK2可能参与发育敏感期视皮层神经元可塑性的调节     

生后发育过程中睾丸神经生长因子基因的表达

为了通过观察小鼠生后发育过程中睾丸组织神经生长因子 ( NGF)基因表达的变化藉以探讨 NGF在睾丸发育、精子发生过程中的生物学作用 ,本研究采用 RT-PCR方法 ,半定量分析生后 1d、1周、2周、4周及 8周小鼠睾丸组织中 NGF m RNA的表达变化 ;以地高辛标记的βNGF c DNA为探针 ,采用原位杂交法观察 NGF m RNA在幼年及成年小鼠睾丸组织中的分布。 RT-PCR结果显示 ,小鼠生后 1d、1周、2周、4周、8周睾丸组织中均有 NGF m RNA的表达 ,以生后 1周时的表达量为最高。原位杂交结果表明 ,幼年鼠 NGF m RNA杂交信号位于睾丸的间质之中 ,而成年鼠 NGF m RNA杂交信号则主要位于睾丸生精小管管壁和管腔中。结果提示 :NGF m RNA在小鼠出生时即有表达 ,其表达量及在睾丸组织中的分布 ,随小鼠的发育而有变化     

Hetrin基因在大鼠脑发育过程中表达的初步研究

本文目的是 :观察 hetrin基因在大鼠脑发育过程中的表达变化规律 ,藉以探讨 hetrin在神经系统发育过程中的作用。以地高辛 (dig)标记 hetrin基因 3 ' -末端 c RNA为探针 ,采用原位杂交法研究 hetrin m RNA在生后第 10 d(P10 )、P3 0和 P60大鼠脑中的分布状况 ;同时采用半定量 RT-PCR法分析出生前后大鼠脑内 hetrin m RNA表达水平的变化。结果发现 ,hetrin m RNA原位杂交阳性信号定位于神经元胞质内 ,在 P10、P3 0和 P60大鼠的脑组织中均可见明显的杂交信号。 RT-PCR结果显示 ,在胚胎第 9d(E9)大鼠脑内 hetrin m RNA开始表达 ,但表达量很低 ,以后其表达量逐渐升高。出生后大鼠不同脑区 hetrin m RNA的表达量变化趋势不同。本实验结果表明 ,在胚胎期及出生后大鼠脑的多个部位都有 hetrin m RNA表达 ,而且 hetrin m RNA表达量与神经细胞突起生长及新突触连接的建立在时间上有一定的同步性 ,提示 hetrin m RNA的表达与神经突起生长存在着一定的关系 ,进一步支持存在 hetrin基因的神经细胞突起具有诱向生长功能     

cAMP反应元件结合蛋白(CREB)在正常发育大鼠视皮层的表达研究

正常视觉信息输入对初期视皮层的发育和修饰至关重要。视觉发育的特点是存在一个关键期 ,在此期内控制信息输入将导致皮质联系的显著变化。转录因子 -c AMP反应元件结合蛋白 ( CREB)在多种有机体的信号传导通路中的枢纽作用和其参与长时程突触可塑性的生理活动提示 ,它可能参与了在视皮层发育中发挥重要作用的分子机制。本研究应用免疫组织化学方法和 Western blot技术 ,对 P14、P2 2、P3 0、P45和 P90年龄组 Sprague-Dawley大鼠视皮层内 CREB的免疫反应性进行观察和分析。结果证明 ,CREB在视皮层各层内的蛋白表达时程与视觉发育的关键期一致 ,其免疫反应性在 P2 2至 P45期间达到高水平 ,成年后的蛋白表达出现下调。结论 :视觉可塑性降低的同时存在 CREB表达的显著改变 ,但该蛋白表达改变与突触传递和可塑性的关系尚待阐明     

cAMP反应元件结合蛋白(CREB)在单眼剥夺弱视大鼠视皮层中的表达研究

目前已对突触可塑性的基本特性有了相当程度的认识 ,但对其发育中神经元可塑性的内在分子机制尚待进一步阐明。不少研究提示 ,c AMP反应元件结合蛋白 ( CREB)在学习、记忆和长时程增强 ( LTP)过程中参与长时程突触的可塑性调节 ,推测其在视觉系统可塑性中也发挥着重要作用。本研究通过建立幼年大鼠单眼剥夺弱视模型 ,应用免疫组织化学方法 ,对视觉发育关键期末 ( P45 )大鼠和成年 ( P90 )大鼠视皮层中 CREB和 p CREB的免疫反应性进行观察、比较和分析。结果 :视觉发育关键期内进行单眼视觉剥夺对大鼠视皮层内总 CREB的蛋白表达没有产生显著影响 ,而 p CREB在 P45大鼠的剥夺眼对侧视皮层单眼反应区的 / 、 层中的蛋白表达较剥夺眼同侧视皮层的相应区域弱 ,该差异在该年龄大鼠的双眼反应区及成年后大鼠视皮层内不存在。结论 :CREB可能通过该磷酸化形式在视觉系统可塑性中发挥作用     

核呼吸因子-1在正常发育和视觉剥夺大鼠视皮层内的表达

目的:观察核呼吸因子-1(NRF-1)在正常发育及视觉剥夺大鼠视皮层内的表达.方法:构建视觉剥夺大鼠模型,利用半定量:PCR、Western blot及免疫组织化学的方法检测NRF-1在正常发育及视觉剥夺大鼠视皮层内的差异表达.结果:通过以上方法均可检测到视觉剥夺大鼠视皮层内NRF-1表达明显降低.结论:NRF-1的基因转录及蛋白表达水平依赖于正常视觉信号的刺激,异常的视觉经验使视皮层神经元兴奋性降低,造成NRF-1表达的明显下调.     

乙型肝炎病毒X蛋白通过激活ERKs和NF-κB上调大鼠系膜细胞表达TNF-α

目的 探讨乙型肝炎病毒(HBV)X基因转染大鼠系膜细胞肿瘤坏死因子-α(TNF-α)高表达的细胞外蛋白调节激酶(ERKs)、核因子κB(NF-κB)、P38 MAPK信号传导机制.方法 将含有HBV X基因的质粒pCI-neo-X导入体外培养的大鼠系膜细胞,分别采用特异性抑制剂U0126阻断ERK1/2通路,Lactacystin阻断NF-κB通路和SB203580阻断P38 MAPK通路,观察培养细胞TNF-α及其mRNA表达,以不加抑制剂作为对照.RT-PCR检测TNF-α mRNA表达,ELISA检测培养上清液中TNF-α表达.Western blot检测乙型肝炎病毒X蛋白(HBx)表达.结果 转染pCI-neo-X后,系膜细胞TNF-α mRNA及上清液TNF-α表达均增高,通过阻断ERK1/2或NF-κB通路,细胞TNF-α mRNA及上清液TNF-α表达均下降.而阻断P38 MAPK通路对系膜细胞TNF-α mRNA及上清液TNF-α表达无明显影响.结论 HBx蛋白通过激活ERKs和NF-κB信号通路使系膜细胞高表达TNF-α,而与P38 MAPK通路无关.     

骨形成蛋白4在大鼠中枢神经系统发育过程中的表达

目的 研究骨形成蛋白4(BMP4)基因在大鼠中枢神经系统(CNS)发育过程中的表达。方法 地高辛标记特异性寡核苷酸探针原位杂交组织化学技术。结果 在E16大鼠，BMP4主要在小脑与嗅球呈强阳性表达。在P1—2大鼠，BMP4 mRNA在延脑的舌下神经核呈强阳性信号，在三叉神经脊束核、脊髓小脑束可见部分BMP4 mRNA阳性细胞。在P1周大鼠，额叶皮层、枕叶皮层与海马的前下托可见较强的阳性信号。生后1个月，海马与皮层BMP4的表达达到生后的峰值。此时BMP4在颞叶皮层与杏仁周皮质呈强阳性表达。生后3个月，BMP4在大鼠CNS有广泛表达，其中在海马、颞叶皮层、杏仁周皮质、丘脑侧核与下丘脑的室旁核均可见强阳性信号，而生后18个月大鼠的皮质、海马、丘脑、下丘脑仍可见一定数目的阳性神经元。结论 提示BMP4对新生期和成年期大鼠CNS的发育起重要作用。     

猫视皮层N-甲基-D-天门冬氨酸受体的基因表达

目的　检测 5种NMDAR亚基的mRNA在猫视皮层的表达情况 ,为NMDAR的功能多样性提供分子基础。方法　 3月龄猫 6只 ,采用地高辛标记的寡核苷酸探针 ,对猫视皮层切片进行原位杂交。结果　NR1的mRNA在视皮层的所有层均有强表达 ,NR2A在Ⅲ、Ⅳ层表达较高 ,而Ⅱ、Ⅴ层呈中等水平 ;NR2B的表达信号较弱 ,主要表达于Ⅰ、Ⅲ和Ⅴ层 ;NR2CmRNA的信号很弱 ,表达Ⅲ、Ⅴ和Ⅵ层 ;NR2DmRNA在视皮层各层呈中等表达 ,以Ⅴ和Ⅵ层的信号最强。结论　NMDAR各亚基在猫视皮层中的基因表达具有多样性 ,这可能构成了不同NR通道具有功能差异的分子基础     

N-甲基-D-天冬氨酸受体NR1亚基调控大脑皮层神经元放射状迁移

目的探讨N-甲基-D-天冬氨酸(NMDA)受体在大脑皮层发育过程中参与调控皮层神经元放射状迁移的情况。方法通过在体子宫内胚胎电转基因和RNA干扰技术(RNAi),在发育的大鼠胚胎脑内干扰NMDA受体的NR1亚基表达,结合荧光免疫组织化学技术,追踪基因转染上的神经元在大脑发育过程中的迁移途径和特征。结果转染上对照质粒的皮层神经元能通过放射状迁移,在出生后第7天(P7)之前迁移到皮层的正确位置,即2～3层,而干扰NR1基因表达后的神经元则不能正常地放射状迁移至皮层的2～3层,P7时仍停留在皮层深层位置,甚至发生区。结论 NMDA受体在大脑皮层神经元放射状迁移过程中起关键性作用,有助于理解胚胎发育过程中迁移的机制并为预防控制神经元迁移异常提供依据。     

猫视皮层ERK mRNA表达的研究

丝裂原激活的蛋白激酶又称细胞外信号调节激酶,具有神经元信号传导功能,参与多种发育过程。它由细胞外特殊信号激活,将酪氨酸磷酸化过程转化为丝氨酸／苏氨酸磷酸化过程,后者进一步调节下游的蛋白激酶。因此,细胞外信号调节激酶在磷酸化的连锁反应过程中起着枢纽作用。本文应用地高辛标记寡核苷酸探针的原位杂交技术,研究了细胞外信号调节激酶的２ 种亚型 ＥＲＫ１ 和ＥＲＫ２ ｍ ＲＮＡ 在生后４ 周龄的幼猫视皮层各层的分布。结果表明,ＥＲＫ１ ｍ ＲＮＡ 在视皮层的表达层次明确,以第Ⅴ层和第Ⅲ层的表达为最强,Ⅱ层呈中等表达,Ⅳ层较弱,Ⅵ层最弱,即在视皮层的表达强度为Ⅴ、Ⅲ＞ Ⅱ＞ Ⅳ＞ Ⅵ。与ＥＲＫ１相比,ＥＲＫ２ 的表达普遍较强,但层次欠清晰,其表达强度为Ⅴ、Ⅱ＞ Ⅲ、Ⅳ＞ Ⅵ。ＥＲＫ 的２ 种亚型在视皮层的Ⅰ层即分子层均不表达。ＥＲＫ１ 和ＥＲＫ２ ｍ ＲＮＡ 在视皮层的这种特征性分布提示,在幼猫的视皮层发育过程中,ＥＲＫ 可能具有独特的信号传递作用。     

Postnatal expression of alpha2 nicotinic acetylcholine receptor subunit mRNA in developing cortex and hippocampus

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of alpha and beta subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of alpha3, alpha5, alpha7, and beta4 subunit mRNAs and alpha7 binding sites during hippocampal and cortical development. Here, we examined the expression of alpha2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. alpha2 Subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of alpha2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of alpha2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, alpha2 mRNA expression was located in non-pyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, alpha2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing alpha2 subunits could regulate GABAergic activity during a critical period of network formation.     

Postnatal expression of α2 nicotinic acetylcholine receptor subunit mRNA in developing cortex and hippocampus

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of α and β subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of α3, α5, α7, and β4 subunit mRNAs and α7 binding sites during hippocampal and cortical development. Here, we examined the expression of α2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. α2 Subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of α2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of α2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, α2 mRNA expression was located in non-pyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, α2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing α2 subunits could regulate GABAergic activity during a critical period of network formation.     

芳香化酶mRNA在小鼠脑内的表达及其分布

目的 研究芳香化酶ｍ（ＲＮＡ（ａｒｏｍａｔａｓｅ ｍＲＮＡ）在小鼠脑内的基因表达。方法 原位杂交组织化学和ＰＮＡ斑点杂交。结果 （１）斑点杂交结果显示,脑内芳香化酶ｍＲＮＡ在小鼠Ｅ１６－Ｐ３００整个发育过程中均有表达,表达高峰在生后６ｄ左右,成年后降至最低;（２）脑内芳香化酶ｍＲＮＡ主要定位于神经元;（３）芳香化酶ｍＲＮＡ在脑内的表达,阳性区域主要分布于大脑皮层,丘脑、下丘脑及边缘系统。其中,皮质锥体细胞层、内侧视前区、隔内侧核、海马各区锥体层、杏仁核群、扣带皮质、梨状前皮质及杏仁周皮质等部位阳性信号较强;中等强度的阳性信号见于丘脑腹内、外侧核,丘脑外侧背核、下丘脑室旁核、室周核等处。结论 以上结果进一步证明脑内芳香化酶的表达与脑发育存在一定的相关性,芳香化酶ｍＲＮＡ的表达部位与文献报道酶的活性分布基本一致;海     

Extracellular signal regulated kinases. Localization of protein and mRNA in the human hippocampal formation in Alzheimer's disease.

MAP kinases (MAPK) are a family of serine/threonine (Ser/Thr) kinases that link cell surface signals to changes in enzyme activity and gene expression. They are the products of the newly described gene family referred to as extracellular signal regulated kinases (ERKs). Moreover, MAPKs phosphorylate tau in vitro at Ser/Thr Proline sites, generating a multiply phosphorylated tau protein that is similar to the hyperphosphorylated tau found in Alzheimer neurofibrillary tangles (NFTs). We studied MAPK immunoreactivity and in situ hybridization patterns of the two major genes that comprise MAPK activity, ERK1 and ERK2, in the human hippocampal formation. Our goal was to determine whether the pattern of ERK expression is consistent with the hypothesis that MAPKs contribute to NFT formation. ERK1 mRNA is present in small amounts and confined primarily to dentate gyrus granule cells. ERK2 mRNA, by contrast, gives a much stronger hybridization signal and is present in dentate gyrus granule cells and pyramidal cells throughout all hippocampal subfields and adjacent temporal neocortex. Quantitative measures of ERK2 mRNA reveal that NFT-bearing neurons contain approximately 15% less ERK2 mRNA than nearest neighbors that do not contain NFT. NFT-bearing neurons contain approximately 25% less polyA mRNA, suggesting a relative preservation of ERK2 mRNA even in metabolically compromised cells. MAPK immunoreactivity (which represents both ERK1 and ERK2) is seen in neuronal soma, dendrites, axons, and in reactive astrocytes. In Alzheimer's disease, neurons that contain NFTs are also MAPK immunoreactive, but neurons that contain the highest amounts of MAPK immunoreactivity are not necessarily vulnerable for NFT. MAPK immunoreactivity is present in the same neurons as NFT and in the same subcellular compartments as tau, supporting a role for MAPKs in tau phosphorylation in Alzheimer's disease. However, the presence of ERK immunoreactivity is not sufficient to predispose neurons to NFT formation.     

Spatial and temporal expression of UDP-galactose: ceramide galactosyl transferase mRNA during rat brain development

 We studied the expression of UDP-galactose: ceramide galactosyl transferase (CGT) mRNA in postnatal rat brains using an in situ hybridization technique. From P0 to P16, there was a defined temporal and spatial pattern to the earliest acquisition of CGT mRNA expression. In the forebrain, CGT mRNA-expressing (CGT⁺) cells were first detected in regions outside the subventricular zone around the lateral ventricle at P2. Cells in the external capsule, internal capsule and corpus callosum were later found to be CGT-positive. At P8 to P16, CGT⁺ cells were found in the thalamus, striatum, occipital and frontal cortex. In the case of midbrain and hindbrain, the first CGT⁺ signals were detected in the medullary raphe of the medulla oblongata at P0. CGT⁺ cells were subsequently located in the cerebellum, midbrain and pons from P4 to P16. That is, in regions closer to the areas in which CGT⁺ cells were first found, CGT mRNA expression was observed much earlier. These findings support the notion that there are at least two discrete waves of CGT mRNA signal expression in the forebrain and hindbrain. Accepted: 3 February 1999     

猫视皮层17区一氧化氮合酶阳性神经元的生后发育

用ＮＡＤＰＨ ｄ 黄递酶组化技术观察了生后不同年龄段猫视皮层１７ 区中一氧化氮合酶阳性神经元的生后发育状况。结果显示： 生后１ 周内,一氧化氮合酶阳性细胞仅出现于皮层的第５／６ 层以及皮质下板（白质）中,皮质板中极少;生后２ 周时,一氧化氮合酶阳性细胞见于２／３ 和４ 层;在随后的发育中,２／３ 层与４ 层中阳性细胞逐渐增多,第５ 周时呈现与成年动物相似的分布模式。一氧化氮合酶阳性细胞的发育模式反映了皮质板的形成过程,即“由内向外”的皮层片层成熟模式;成年动物的皮层１７ 区中一氧化氮合酶阳性细胞远远高于幼年。猫视皮层１７ 区中一氧化氮合酶阳性细胞发育的时空表达模式与猫视皮层发育的关键期无明显吻合关系,推测视皮层中内源性一氧化氮与视皮层的眼优势柱的可塑性无关,为Ｒｅｉｄ 等和Ｒｕｔｈａｚｅｒ 等的电生理学研究结果提供了形态学证据。