【作者】 申军现；

【导师】 鞠躬；

【作者基本信息】 中国人民解放军第四军医大学 ， 神经生物学， 2003， 硕士

【摘要】 目前认为CNS轴突不能再生的主要因素之一是CNS内存在众多的抑制因子，髓鞘是抑制因子的主要来源。近年来认为髓鞘表达的NI-250和NI-35蛋白可能是CNS轴突再生失败的主要因素，用NI-250作为免疫原制备的单克隆抗体IN-1可识别NI-250和NI-35，IN-1的体内研究结果奠定了NI-250和NI-35作为中枢神经再生中最重要的抑制因子的地位。体内应用IN—1可以诱导损伤的纤维长距离地延伸，并可引起某些特定的反射、运动功能的恢复。通过对NI-250分子的六个肽序列的微测序，2000年克隆了nogo基因，Nogo分子有三种不同的剪接体—Nogo-A，-B，-C，其中Nogo-A对应于NI-250，NI-35可能为NI-250的降解片断。三种Nogo分子都具有相同的188个氨基酸的C-末端，与Reticulon家族成员高度同源。在Nogo分子的C-末端，有两个跨膜片段，被66个氨基酸残基组成的环状结构(Nogo-66)所分隔。Nogo-66的受体分子—NgR于2001年随之被克隆。NSR是一个带有LRR(leucine rich repeat)结构域的GPI(glycophosphatidylinositol)膜锚定蛋白，介导Nogo-66抑制轴突生长的作用。在体外培养系统中，Nogo-A分子的酸性氨基端和Nogo-66片断都 第四军医大学硕士研究生论文一表现出强的抑制突起生长的活性，提示该分子至少具有两个不同的抑制性结构域。 Northern Blot、原位杂交、Western Blot和免疫组化结果证明：Nogo－AmRNA和 Nogo蛋白除了在 CNS的寡突胶质细胞中表达，还表达于发育阶段和成年的脑、脊髓和外周神经节的某些神经元中，在外周组织如睾丸和心脏也有表达；NOgOE在CNS和PNS以及多种外周组织中有广泛分布；NOgO（除表达于脑和心脏外，在骨骼肌中有较高表达。NOgO人在别的组织，尤其是在神经元中的广泛表达，以及NOgO羽和NOgO（的广泛分布，表明生理状态下，NOgO分子不仅仅是扮演抑制因子的角色，可能具有更多重要的功能不被人们认识。 为探讨NOgO－A在神经元中的表达与神经元发育分化的关系，本研究借助了海马神经元常规培养方法和低密度条件下培养方法，应用针对Nogo－A的特异性抗体，采用免疫荧光细胞化学染色和 Western Blot方法，观察了NOgO－A在体外培养的大鼠海马神经元表达分布模式，和不同培养时间NOgO－A亚细胞分布的变化。并培养了大鼠胶质细胞和PC 12、3T3等细胞作为对照。结果提示NOgO－A在不同培养时间海马神经元中均有表达，主要分布在胞浆、胞膜和突起上。神经元突起形成过程中，NOgO－A主要表达于树突近端，在轴突上随着轴突的伸长逐渐表达于轴突远端和生长锥。NOgO－A在成熟的神经元网状的突起上，呈串珠样分布，这些结构推测为膨体或生长锥。以上结果提示NOgO－A在神经元中具有不同于抑制作用的其他功能，可能参与神经元突起生长、突触形成等分化过程。更多还原

【Abstract】 One of the major reasons of the failure of CNS axon regeneration is the presence of many inhibitory molecules, mostly originated from CNS myelin. Nogo protein expressed by CNS myelin is regarded as a major repulsive factor for the failure of CNS axon regeneration. The interest in this protein stems principally from the neurite growth promoting effect of a monoclonal antibody (IN-1) rasied against NI-250 which has been proved later to be a part of Nogo-A. In vivo, this function-blocking antibody has been shown to partially neutralize the growth inhibitory effect of CNS myelin, and induce long-distance fiber regeneration in spinal cord injuries of the adult mammalian CNS with a recovery of specific reflex and locomotor functions. The cloning of nogo gene in 2000 provides a fundamental tool to comprehensively study the structure and function of Nogo protein. Three major transcipts (Nogo-A, -B, -C)originate from the nogo gene by alternative splicing. The three isoforms have a common carboxy-terminal domain of 188 amino acids, and this region is highly homologous to the reticulon protein family. The carboxyl portion of the Nogo protein contains two transmembrane domains, which are seperated by an extracellular 66-amino-acid fragment (Nogo-66). Nogo-66 receptor, NgR, cloned in 2001, is a leucine-rich-repeat glycophosphatidylinositol-anchored membrane protein which mediates Nogo-66 inhibition of axonal outgrowth. Both the long acidic amino-terminal domain and the Nogo-66 fragment have strong neurite growth inhibitory activity suggest that Nogo-A has at least two inhibitory domains.Northern blot, in situ hybridization, Western blot and immunocytochemistry analyses show that in addition to oligodendrocytes, Nogo-A mRNA and Nogo-A protein are also expressed in neurons in developing and adult brain and spinal cord, Nogo-A is also found in peripheral organs such as heart and testis. Nogo-B is widely distributed in CNS, PNS and many peripheral tissues. Nogo-C is expressed in brain, heart, and skeletal muscle. The wide expression of Nogo-A, -B and -C suggests that the Nogo family proteins may have function(s) in addition to neurite growth inhibition.To investigate the potential function of Nogo-A involved in neuronal development and differentiation, this study mainly used hippocampal neuron culture model, both in conventional and low-density condition, and immunohistochernical and Western blot techniques. Nogo-A expression pattern in hippocampal neurons at different stages and its subcellular distribution were explored by using specific antibody raised against Nogo-A. PC12, 3T3 cells were also cultured as controls. Nogo-A was found in hippocampal neurons atdifferent stages and is located in cytoplasm, membrane and neurites. Nogo-A was detected at proximal part of all neurites before axon formation. Then, Nogo-A expression was gradually enriched in the distal segment and growth cone of the putative axon which grew much faster than other neurites. In mature neuronal network, Nogo-A immunoreactive axons showed a string of beads which correspond to the varicosities or growth cones. The results indicate that Nogo-A expressed in neuron may have function(s) in addition to the neurite growth inhibitory activity, and Nogo-A may play a role in the course of neuronal differentiation such as neurite growth and synapse formation.更多还原