【作者】 宁群；

【导师】 舒斯云；

【作者基本信息】 第一军医大学 ， 细胞生物学， 2002， 博士

【摘要】 边缘系统是指中枢神经一组多相结构的组合，这些结构包括扣带回、旁海马回、杏仁复合体、海马结构、隔核、丘脑前核、内侧核和部分基底节。该系统涉及各种内脏和躯体功能的控制以及辨认、情绪、动机、学习记忆等行为。边缘系统这一概念广泛应用于基础和临床的神经解剖学领域，但关于其神经结构的组成目前仍有争议。近年来新技术的应用发现边缘系统环路涉及中枢神经系统的区域越来越广泛，因此神经科学家试图寻找边缘系统的共同特征。现在已发现一种糖蛋白，主要在边缘系统结构的神经元胞体表面、轴突膜和生长锥中表达，命名为边缘系统相关膜蛋白，被认为是边缘系统共同的分子标志。 边缘区(Marginal division,MrD)是舒斯云在哺乳动物纹状体内发现的一个新亚区。该区域位于纹状体内尾壳核和苍白球之间，为由梭型细胞组成的，呈腹背方向走行的带状结构。因其位置和形状而命名为边缘区。其细胞形态、纤维联系、所含的神经递质及功能均不同于纹状体的其它部分。既往研究表明边缘区参与了学习记忆过程，而学习记忆被认为是脑边缘系统的重要功能；刺激边缘区后，发现边缘系统的一些结构有原癌基因的表达；同时纹状体边缘区与Meynert基核，丘脑及黑质网状部、苍白球内侧有纤维联系。这些资料均提示纹状体边缘区可能与边缘系统有关联。因此为进一步确证纹状体边缘区与边缘系统的关系，以明确边缘区是否属于边缘系统的一个组成部分，本课题的第一部分在以下三个方面进行了研究：1．检测了边缘系统标志分子—边缘系统相关膜蛋白在边缘区的表达；2．研究了边缘区与边缘系统的功能联系；3．研究了边缘区与边缘系统的纤维联系。 大脑可分为多个脑区，每个脑区的功能、联系均不同。不同的基因表达在决定脑区的定位和内在的特质中起重要作用。边缘区在细胞形态、纤维联系和功能方面均有其特异性，推测其基因表达也有其特殊性，因此课题的第二部分对边缘区特异基因的筛选做了初步探索。 方洁和结果 第一部分：l．边缘系统相关膜蛋白在边缘区及相关脑区的表达：成年的SD大鼠，深度麻醉后取出大脑，从中切取海马、尾壳核、边缘区、苍白球、前额叶、小脑等脑区，用 Western blot和 RTICR方法检测边缘系统相关膜蛋白在上述脑区的表达。Western blot显示一分子量为67KD的蛋白条带。边缘区、海马、苍白球和前额叶皮层蛋白带表达较浓，而尾壳核的蛋白带表达较弱，小脑则未见阳性蛋白带表达。RT－PCR检测发现一 200 hp条带，1’－－xrnA表达强度与蛋白表达基本一致。对其进行光密度分析，发现 LA：Nll， InRNA在边缘区的表达强度与海马一致，明显高于尾壳核、小脑。2边缘区与边缘系统的功能联系：用神经系统功能活动形态定位法中的C－fOS表达法研究边缘系统中与学习记忆有关的重要脑区海马、杏仁核与边缘区的功能联系。001％的海人藻酸分别注射到海马和中央杏仁核作为化学刺激剂，用免疫组化法检测c．fos在脑内的表达分布。结果发现刺激海马后，C－fOS主要表达在中央杏仁核、皮层、丘脑部分核团；在纹状体中，c．fos的表达集中在边缘区。刺激杏仁核后，c．fos主要表达在海马、皮层、丘脑部分核团等脑区；在纹状体中，边缘区的。fos表达明显高于尾壳核，而苍白球几乎无c－fos阳性表达。3 边缘区与边缘系统的纤维联系：用霍乱毒素B．辣根过氧化物酶混合顺行和逆行追踪方法研究边缘系统中杏仁核与边缘区的纤维联系；用免疫细胞化学方法观察P物质纤维在边缘区和杏仁核的分布和联系。结果发现：在海马、前梨状皮层可见标记的神经元胞体，颖叶听区及纹状皮层有标记的纤维。在纹状体中，边缘区可见标记的胞体和纤维，胞体为梭型，呈带状分布。在纹状体的尾壳核可见到标记的纤维呈斑片状分布。丘脑中终纹床核、丘脑腹外侧核等可见到标记的胞体。SP阳性纤维集中分布在尾壳核和苍白球之间的边缘区，纤维呈腹背方向走行，呈 — —4—— 带状分布。在杏仁核中SP物质主要集中在中央杏仁核和内侧杏仁核。 在终纹床核内也可见到SP阳性纤维。边缘区的SP阳性纤维在背侧与终 纹床核相连，在腹侧延续至杏仁核。 第二部分：边缘区及相关脑区基因表达差示分析的初步研究：引产 胎脑，取出海马、杏仁核、边缘区和尾壳核。用InRNA差异显示技术 研究各脑区的基因表达，发现各脑区的基因表达都不相同，比较各脑区 的基因表达，发现边缘区有2条差异序列（序列1，2），一条只与尾 壳核共有的序列（序列3）；海马有两条差异序列。对序列1，2，3和 海马的一条差示序列（序列4）测序并进行了生物信息学分析，发现序 列1，4在己知基因中未见同源序列，序列2为原癌基因Dek内含子的 一部分，序列3为酪氨酸激酶的下游序列。对序列1和4进更多还原

【Abstract】 PurposesThe term limbic system refers to a heterogeneous collection of structures in the central nervous system. This system includes the cingulate and parahippocampal cortex, the amygdaloid nuclear complex, the hippocampal formation, the septal nuclei, the anterior and midline thalamic nuclei and parts of the basal ganglia. These anatomically connected areas of the brain are physically associated with the maintenance of various visceral and somatic functions related to discriminative, motivational and emotional behaviors and are involved in learning and memory. Although the term "limbic system" is commonly used in basic and clinical neuroanatomical descriptions, there is lack of agreement regarding the neural structures that it comprises. Recent years, studies with some new techniques suggested that limbic circuitry involves multiple areas of the central nervous system, so the search for common characteristics of the limbic system has become an attractive strategy for neurobiologists. Now a type of glycoprotein has been found selectively expressed on the surface of cell bodies, axonal membranes, and growth cones of the neurons in the limbic system. It is termed limbic system-associated membrane protein (LAMP), and regarded as the common molecular marker of the limbic system.The marginal division (MrD), first discovered by Shu Siyun, is a new subterritory of striatum with function related to learning and memory. It forms a belt-shaped zone locating at the caudal margin of the striatum and surrounding the globus pallidus, running dorsoventrally. Based on its location and shape, the area is termed "marginal division". It is distinguished from the rest of striatum by the neuron shape, special connections, function, and intensely expressed immunoreactivities of many neuropeptides andneurotransmitters. Studies in the past years showed that MrD has fiber connection with the reticular part of substantia nigra, the medial part of globus pallidus, Meynert basal nucleus and interior nucleus of posterior plate in thalamus. The oncogene c-fos expressed in some of the limbic structures following stimulating the MrD. MrD also possessed some functions of limbic system, such as learning and memory. All the data suggest the MrD in striatum maybe has some relation to the limbic system. Therefore, in order to further study the relationship between the limbic system and marginal division, and identify whether the MrD belongs to the limbic system, the stress was put on the study about following 3 aspects: (1) the expression of the LAMP in MrD; (2) the functional connection between the limbic system and MrD; (3) the fiber connection between the limbic system and MrD.The brain is divided into numerous regions. Each brain region is characterized by its unique function, connection and architecture. The differential gene expression has primary role in regulating arealization and is established by mechanisms intrinsic to it. The MrD has also the specificity in connection, function and neuron shape, it must have specific gene expression. Thus, preliminary studies on investigating specific candidate genes of the MrD have also been done in this study.Methods and ResultsPart one: 1. The LAMP expression in the MrD and related brain regions: Adult SD rats were deeply anaesthetized with chloral hydrate. The brain was exposed. The MrD, caudate putamen, globus pallidus, prefrontal cortex, hippocampus and cerebellum were precisely dissected under a macroscope. The Western blot and RT-PCR techniques were used to investigate the expression of LAMP and LAMP transcript respectively. The Western blot revealed a specific protein bands at molecular weights of 67KD, the bands from protein extracted from the MrD, hippocampus, globus pallidus and prefrontal cortex were much darker than that from the caudate putamen, and no positive band was detected from the cerebellum. A 200bp band was shown by RT-PCR. The expression of LAMP mRNA was consistent with the protein. The analysis of semi-quantity suggested the expression of LAMP mRNA in MrD was更多还原