脊髓神经元中微管相关蛋白—5的分布及可塑性

用新生Wistar大鼠进行脊髓神经元培养,研究微管相关蛋白-5与其单克隆抗体结合后的分布情况,使用微管解聚药nocodazole处理神经元,应用免疫组织化学染色来观察微管相关蛋白-5的改变,另一组神经元使用nocodazole处理后再用PMA处理,观察微管相关蛋白-5及神经元的改变。结果表明,微管相关蛋白-5在胞浆及突起中均有分布,并随着培养天数的递增而递减。使用nocodazole后神经元中微管相关蛋白-5的分布及数量明显减少。PMA处理神经元后尽管使微管相关蛋白-5的正常结构被破坏,而神经元的伸展却不受影响。     

原代培养脊髓神经元线状溶酶体与细胞骨架蛋白-纽蛋白关系的研究

目的研究原代培养脊髓神经元线状溶酶体(nematolysosome)的形成与分布及其与细胞骨架蛋白-纽蛋白(vinculin)的关系.方法用细胞松弛素D(cytochalasin D,CD)及佛波醇酯(phorbol myristate acetate,PMA)处理原代培养脊髓神经元,用免疫荧光双标记纽蛋白及组织蛋白酶D(cathepsin D)、酸性磷酸酶(ACPase)、电镜细胞化学及共焦激光扫描显微镜方法研究线状溶酶体与纽蛋白的关系.结果在正常对照组神经元,组织蛋白酶D(标记溶酶体)与纽蛋白分布于胞质及突起内;在CD及PMA处理神经元,纽蛋白及组织蛋白酶D的分布呈向心性移动,但集聚的部位不同;电镜酶细胞化学方法显示CD组及PMA组神经元内线状溶酶体均增多.结论组织蛋白酶D及纽蛋白在培养脊髓神经元内协同分布,CD及PMA均可引起二者分布的变化,提示纽蛋白可通过增强细胞内吞体/溶酶体系统活动而使线状溶酶体增加,也可通过促进丝状肌动蛋白聚合而影响线状溶酶体的形成及运动.     

玉米根细胞中类整合素蛋白与α-微管蛋白的共定位及其可能的相互作用

采用间接免疫荧光标记法对玉米根细胞中的类整合素蛋白和细胞骨架主要组分之一的α-微管蛋白进行了荧光定位。结果表明：类整合素蛋白主要分布在质膜上。与对照相比,用与类整合素蛋白特异结合的5肽GRGDS处理后,质膜上类整合素的分布更为均匀,微管的排列密度降低,而用不与类整合素蛋白特异结合的GRGDS类似物SDGRG处理则对类整合素蛋白分布和微管蛋白的排列均无明显影响。微管蛋白解聚剂或稳定剂处理改变类整合素在质膜上的分布。这些结果表明类整合素蛋白与微管蛋白间有复杂的相互作用。     

纽蛋白在原代培养脊髓神经元的分布及其与神经元形态的相关性

目的　用免疫组织化学及形态学等方法对原代培养大鼠脊髓神经元的形态及其纽蛋白 (vinculin)分布进行研究。方法　实验采用原代培养的大鼠脊髓神经元 ,用细胞松弛素 D(cytochalasin D) -丝状肌动蛋白解聚剂处理细胞后 ,用相差显微镜观察细胞形态 ,同时用单克隆抗体免疫组化方法显示细胞内纽蛋白的分布。结果　原代培养的脊髓神经元可见 2～4个细长的突起 ;免疫组织化学方法显示纽蛋白在神经元的胞体及突起均有分布。细胞松弛素 D处理细胞后 ,神经元胞体变大 ,轮廊不清 ,突起增多 ,变短、变粗 ,多分支且分支末端膨大 ;免疫组织化学方法显示纽蛋白在核周的分布明显增加 ,而在突起内的分布则变得不连续 ,呈散在点状。结论　丝状肌动蛋白 (filem ental- actin,F- actin)的完整性对维持神经元的正常形态是必需的 ;神经元形态的变化与纽蛋白分布的变化相关     

神经干细胞分化过程中微管蛋白表达变化的光电镜观察

目的对神经干细胞向神经元定向分化过程中微管蛋白的表达变化进行光、电镜观察研究。方法采用细胞培养技术、免疫荧光技术以及免疫电镜技术对神经干细胞向神经元定向分化过程中微管蛋白的表达变化进行观察。结果在神经干细胞向神经元定向分化的不同时期,存在微管蛋白的表达变化,在分化初期以核周附近分布明显,随神经元的成熟散在分布于胞质中及突起内,形成细网状,构成细胞骨架,维持细胞形态。结论在神经干细胞向神经元定向分化过程中伴随有微管蛋白的表达变化,随神经元的成熟而构成细胞骨架,维持细胞形态。     

纽蛋白在原代培养脊髓神经元的分布及其在神经元形态的相关性

目的：用免疫组织化学及形态学等方法对原代培养大鼠脊神经元的形态及其纽蛋白（vinculin)分布进行研究。方法：实验采用原代培养的大鼠脊髓神经元,用细胞松驰素D（cytochalasinD)－丝状肌动蛋白解剖处理细胞后,用相差显微镜观察细胞形态,同时用单克隆抗体免疫组化方法显示细胞内纽蛋白的分布,结果：原代培养的脊髓神经元可见2－4个细长的突起,免疫组织化学方法显示纽蛋白姑神经元的胞体及突起均有分布,细胞松驰素D处理细胞后,神经元胞体变大,轮廓不清,突起增多,变矩,变粗,多分支且分支末端膨大,免疫组织化学方法显示纽蛋白在核周的人布明显增加,而且在突起内的分布则变得不连续,呈散在点状。结论：丝状肌动蛋白（filemental-actin,F-actin）的完整性对维持神经元的正常形态是必需的,神经元形态的变化与纽蛋白分布的变化相关。     

PKC对原代培养神经元NF-кB表达的影响

用蛋白激酶C（PKC）刺激剂佛波醇酯（PMA）和PKC抑制剂Rottlerin处理培养神经元,RT—PCR法检测神经元NF—xBp65 mRNA的表达;用PMA和NF—κB抑制剂TPCK处理培养神经元,流式细胞术AnnexinV／PI法检测细胞早期凋亡率。观察PKC对原代培养神经元核转录因子kappaB（NF～KB）表达的影响,探讨PKC参与缺血性神经元损伤的可能机制。结果显示PKC可促进NF—κB的表达;NF—κB可诱导培养神经元的早期凋亡。由此可以得出PKC激活参与神经元的损伤可能是通过引起神经元内NF—κB的表达而实现的.     

神经元微管蛋白的研究进展

神经元特殊形态的形成及维持主要依赖于神经元细胞骨架中微管的装配,在此过程中,涉及到微管的组成及其动力学性质,而最终形成了稳定的微管结构,在神经元中,这一结构为沿着神经突运输物质提供了基础。本文将主要在神经元微管的结构与功能,神经元微管蛋白异构基因的表达及其翻译后加工形式等方面的研究进展加以综述。     

6-DMAP诱导PM胚胎形成过程中核纤层蛋白B的动力学变化

我们的前期研究发现:被微管抑制剂nocodazole抑制在第一次有丝分裂中期的小鼠受精卵在加入6-DMAP处理后核膜重新出现,并且父、母本的基因组未发生融合,分别形成了类似雌、雄原核的两个细胞核,它们共存于卵细胞质中,我们把这种特殊的胚胎称之为PM胚胎(post-mitoticembryo)。本研究表明:在去除抑制剂3h后未能形成核膜的胚胎进一步卵裂,而形成核膜的PM胚胎培养24h未见进一步发育。此外,我们采用免疫荧光染色观察PM胚胎核膜重现过程中核纤层蛋白B的动力学变化,结果显示:在加入6-DMAP后核纤层蛋白B在染色体周围逐渐聚集,约3h后核膜完全形成,此时核纤层蛋白B在染色体周围的聚集达到最高峰。文中还对6-DMAP诱导核膜形成的机制进行了探讨。     

嗜热四膜虫接合生殖后期皮层细胞骨架蛋白的研究

用秋水仙素和细胞松驰素B处理接合期的嗜热四膜虫,以观察其对接合生殖期,尤其是接合后期的嗜热四膜虫皮层细胞骨架蛋白的影响,用秋水仙素处理的试验组的皮层细胞骨架蛋白组分中34KD、37KD、46KD和57KD蛋白的含量有明显改变,而用细胞松驰素B处理的试验组中40KD和74KD蛋白的含量改变较大。根据相关文献,作者推测34KD、37KD、46KD、57KD蛋白是微管蛋白,而40KD和74KD可能是微丝蛋白。这些蛋白对嗜热四膜虫接合过程中的形态发生的重要作用有等进一步研究。     

GDNF对原代培养脊髓神经元的影响

本文研究了GDNF对体外培养各个时期的脊髓神经元的作用。通过MTT法检测GDNF对脊髓神经元存活率的影响,发现GDNF能促进培养7天及14天的神经元存活。 通过活体观察、尼氏染色、NSE免疫细胞化学染色观察GDNF对脊髓神经元生长锥数目、胞体大小、突起长度及分枝、侧棘形成的影响,发现GDNF对体外培养1—3周的脊髓神经元有明显的营养作用。     

Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile- treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'- Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.     

THE NUCLEAR DNA CONTENT OF LARGE VENTRAL SPINAL NEURONS

Abstract— A nuclear fraction has been obtained from large spinal neurons previously isolated from bovine ventral spinal cord in bulk suspensions. The fraction contained an average of 5.3 ± 0.9 pg DNA/nucleus, indicating a high incidence of diploid nuclei. This conclusion was confirmed by distribution analysis of DNA in propidium iodide-stained nuclei examined by flow microfluorometry. That technique showed that at least 90% of the nuclei from large spinal neurons are diploid. Mixed, mostly non-neuronal nuclei derived from many types of cells in the ventral spinal cord contained an average of 5.9 ± 0.6 pg DNA/nucleus, 19% of which possibly possess more than diploid amounts of DNA. The uniform DNA content in nuclei of large spinal neurons and most other types of cells in the ventral spinal cord contrasts sharply with a wide variation (av 26-fold) in the nuclear volumes of the same cells.     

MAP 4: occurrence in mouse tissues

A polyclonal antiserum to a microtubule-associated protein (MAP) from mouse neuroblastoma cells (MAP 4) was used to examine the distribution of this protein in mouse tissues. Immunoblots of neuroblastoma cell microtubule protein preparations demonstrated that the antiserum reacted with a triplet of proteins at 215,000-240,000 mol wt. Antibodies affinity purified from any of the bands showed cross- reaction with the other bands, indicating these polypeptides were all immunologically related. Antibodies specific to MAP 4 decorated microtubules in cultured murine cells fixed with glutaraldehyde, and diffuse staining was seen following treatment of cells with nocodazole. The antiserum reacted with MAP 4 in extracts of brain, heart, liver, and lung from adult mouse; the triplet in brain was more closely spaced than in the other tissues or neuroblastoma cells. In kidney, spleen, and stomach, only a single band (band 4) was labeled; this band was immunologically related to the triplet and was also present in all tissues positive for the triplet. Skeletal muscle, sperm, and peripheral blood contained no reactive polypeptides. After taxol- induced polymerization, the MAP 4 triplet was preferentially associated with the microtubule pellet whereas band 4 remained in the supernatant. These data indicate that there is tissue specificity in the distribution of MAP 4, and that some tissues contain a polypeptide related to MAP 4 (band 4) that does not bind to microtubules in vitro.     

DEVELOPMENT OF THE BLOOD-BRAIN BARRIER FOR 6-HYDROXYDOPAMINE

The postnatal development of the blood-brain barrier for the neurotoxic action of 6-hydroxydopamine on central noradrenaline neurons has been investigated by recording the in vitro uptake of [³H]noradrenaline in slices from cerebral cortex, hypothalamus and spinal cord in rats treated with large doses of 6-hydroxydopamine at different ages. The [³H]noradranaline uptake was permanently and markedly reduced in all regions when the animals were treated at birth, certainly related to degeneration of noradrenaline neurons, caused by 6-OH-DA. In the cerebral cortex and hypothalamus an efficient protection against the effects of 6-OH-DA on [³H]noradrenaline uptake developed postnatally, while in the spinal cord this protection was never seen to become complete. The results obtained indicate a rapid formation of a blood-brain barrier for 6-OH-DA in the cerebral cortex between the 7th and 9th day after birth. In the hypothalamus the development of this barrier seemed to have a more gradual time-course, but appeared to be fully developed already at day 5 postnatally. Also in the spinal cord the barrier developed more gradually from birth to the adult age. It was observed, however, that both in the cerebral cortex and in the spinal cord, the blood-brain barrier developed, could not completely protect the central noradrenaline neurons from the neurotoxic actions of large doses of 6-OH-DA administered systemically to adult rats. Furthermore, the results obtained support the view that 6-OH-DA does not seem to apparently affect the outgrowth of remaining NA neurons which have not been destroyed by the 6-OH-DA treatment.     

离体培养的小鼠脊髓固有神经元的突触构筑

运用电子显微镜观察分析原代分离培养鼠胚脊髓的固有神经元的突触构筑。培养中主要可见中、小型神经元,彼此之间可形成大量的突触,以非对称性突触占多数,有轴-树突触和轴-体突触,树-树突触为少见。根据以前学者分类标准将终扣分成S、F、M、和G四型。超微结构有利于提示固有神经元经过简单突触从脊髓固有神经纤维接受突触传入,表示它们的冲动只是突触后机制控制信息传递。     

Reelin promotes microtubule dynamics in processes of developing neurons

The extracellular matrix protein reelin controls radial migration and layer formation of cortical neurons, in part by modulation of cytoskeletal dynamics. A stabilizing effect of reelin on the actin cytoskeleton has been described recently. However, it is poorly understood how reelin modulates microtubule dynamics. Here, we provide evidence that reelin increases microtubule assembly. This effect is mediated, at least in part, by promoting microtubule plus end dynamics in processes of developing neurons. Thus, we treated primary neuronal cultures with nocodazole to disrupt microtubules. After nocodazole washout, we found microtubule reassembly to be accelerated in the presence of reelin. Moreover, we show that reelin treatment promoted the formation of microtubule plus end binding protein 3 (EB3) comets in developing dendrites, and that EB3 immunostaining in the developing wild-type neocortex is most intense in the reelin-rich marginal zone where leading processes of radially migrating neurons project to. This characteristic EB3 staining pattern was absent in reeler. Also reassembly of nocodazole-dispersed dendritic Golgi apparati, which are closely associated to microtubules, was accelerated by reelin treatment, though with a substantially slower time course when compared to microtubule reassembly. In support of our in vitro results, we found that the subcellular distribution of α-tubulin and acetylated tubulin in reeler cortical sections differed from wild-type and from mice lacking the very low density lipoprotein receptor (VLDLR), known to bind reelin. Taken together, our results suggest that reelin promotes microtubule assembly, at least in part, by increasing microtubule plus end dynamics.