【作者】 石军；

【导师】 蔡文琴；

【作者基本信息】 第三军医大学 ， 组织学与胚胎学， 2001， 博士

【摘要】 多巴胺（dopamine，DA，化学名3，4-羟基苯丙胺）是中枢神经系统主要的儿茶酚胺类神经递质，多巴胺参与控制机体运动、行为、情绪、摄食及内分泌调节等。在中枢神经系统所有的慢作用递质中，DA是长期以来受到最多关注的神经递质，主要原因是其与神经系统重大医学问题联系最为紧密。临床上常见疾病如帕金森氏病（Parkinson’s disease，PD）是中脑黑质DA能神经元的选择性退变所致，亨廷顿氏舞蹈病（Huntington’s chorea）是由于纹状体内DA投射神经元的损害，目前所有的精神分裂症（schizophrenia）治疗药物在本质上主要是以DA受体的拮抗剂起作用，注意力缺陷/过敏障碍（attention deficit hyperactivity disorder，ADHD）的症状可用DA能神经调节药物得到极大缓解。药物可卡因、苯丙胺、阿片与尼古丁、酒精等通过调节DA神经传递而获得成瘾性。人们已越来越清楚地认识到，DA在人类身心健康中具有非常重要的生理作用。DA的研究也成为了当今神经科学中的前沿研究领域，为揭示人类思维和精神活动规律及其有关疾病的奥秘提供了可能的途径。 尽管人们对DA的研究有广泛的兴趣，并且也取得了辉煌的成就，但目前对DA发生作用的细胞和分子机制仍不清楚。随着近年来细胞或分子水平研究的广泛开展，发现脑内除神经元以外胶质细胞也是DA作用的重要靶细胞。事实上，脑组织主要由神经元和胶质细胞共同构成，数量上胶质细胞与神经元比是10～50：1，胶质细胞占脑体积一半以上，已经表明胶质细胞在神经系统具有非常重要的功能。最早的实验发现脑组织胶质细胞的提取物有腺甘酸环化酶相关的DA受体，接着，发现DA可刺激皮质和纹状体，非脑干来源的培养星形胶质细胞CAMP积聚。进一步应用电生理和放射性配体结合自显影研究提示胶质细胞具有DA受体。我们应用pH］DA放射性受体分析法（r。山。reCeptofN11山ng SSS。y，R RARA）研究发现三个不同部位来源的星形胶质细胞具有5个不同的解离常数，表明胶质细胞分布广泛而且具有部位异质特性。近期的研究利用分子生物学技术如PCR和原位杂交，证实培养纹状体胶质细胞表达DI和DZ受体。另外，胶质细胞具有类似神经元的DA摄取系统。由此可见，胶质细胞是脑内DA作用的重要靶细胞，并且胶质细胞可能在DA神经系统疾病中具有非常重要的作用。本研究利用细胞与分子生物学的多种技术方法，克隆与鉴定系列胶质细胞DA的相关基因，并对部分新基因功能进行初步的特性分析。一方面，从理论上有利于分析DA作用于星形胶质细胞的分子机制，了解胶质细胞在＊A神经系统中的生理功能：另一方面，可能获得＊A系统疾病的相关基因，对揭示帕金森氏病，精神分裂症和药物成瘸等疾病的病因，提高临床诊断以及治疗水平有重要的应用价值。 本研究首先从大鼠脑组织分离培养星形胶质细胞，以多巴胺DA处理后提取 mMx，然后结合应用抑制消减杂交侣u以ession subtractlvehybridization，SSH）和选择性差异筛选（PCR·Select Differential Screening）方法，分离获得27个DA调节表达cDNA克隆。经Northern杂交验证，序列测定与生物信息学分析，明确了 14个 DA调节表达的基因，包括神经递质转运相关的多功能调节蛋白泛素蛋白连接酶Nedd4，调节细胞分泌活动的内质网钙联结蛋白，能量代谢相关的葡萄糖调节蛋白，DA代谢相关酶NAD（P）H－酮还原酶和NADH－泛酮氧化还原酶，以及铁蛋白H亚单位等。实验结果提示＊pA激活了胶质细胞内复杂的信号传递通路，并且涉及生长因子信号途径、凿体激素信号途径和白介素调节通路之间的相互交流（cros叶alking卜（）信号传递的靶基因有四类：代谢酶，应激蛋白，转运蛋白，以及生长发育调节蛋白等。O）其中几个基因变异与神经疾病密切相关，也提示多巴胺能神经疾病与非多巴胺能神经疾病之间也有某些共同的机制。 Vlll 人类基因的发现及功能研究，尤其重要疾病的相关基因研究是目前世界范围的重大课题。多巴胺＊opamine，DA）是中枢神经系统重要的神经递质，与神经系统重大医学问题联系最为紧密。胶质细胞DA相关基因的克隆对揭示胶质细胞在DA系统中的重要作用，以及对揭示帕金森病，精神分裂症和药物成噶等DA系统疾病的病因，提高临床诊断以及治疗水平有重要的应用价值。本研究采用同源克隆策略川 CIOllillg），采用DA处理的大鼠星形胶质细胞的消减PCR探针，筛选人类胎脑cDNA噬菌体文库。通过高质量全长cDNA噬菌体文库的构建，经过严格的杂交的筛选，挑选了96个阳性克隆，经过测序分析明确了61条DA相关的全长基因，并且筛选到数个全长的新基因。通过文献查阅与生物信息学分析，比较全面地了解了DA激活星形胶质细胞的信号传递机制及其相关功能意义，并且新基因的克隆为进一步的基因功能研究及其开发应用研究奠定了重要的基础。 在我们筛选到的数条新的人类全长cDNA基因中，依据生物信息学分析我们选择两更多还原

【Abstract】 Molecular Cloning and Characterization of DopamineResponsive Genes From AstrocytesAbstractDopamine (3,4-dihydroxyphenethylamine; DA) is the predominant catecholamine neurotransmjtter in the mammalian central nervous system. DA controls a variety of functions including locomotor activity, cognition, emotion, positive reinforcement, food intake, and endocrine regulation. Of many slow-acting neurotransmitters, DA has received by far the most attention, mainly because that several pathological conditions have been linked to dysregulation of dopaminergic transmission. It has been found by a number of investigators that glial cells are targets of DA. However, few studies have reported on the molecular cascade associated with receptor activation and DA metabolism in glial cells during dopaminergic neuronal activity.In this study, a series of cellular and molecular biological technichies were applied to isolate and identify the DA responsive genes from glial cells.Analysis of DA responsive genes should provides a better understanding of the functional impact of glial cells in dopaminergic transmission, and the complex cellular and molecular mechanism of the disorder of dopaminergic transmission including Parkinson’s disease, schizophrenia, and drug addiction etc.In the present study, we have investigated the influence of DA on gene transcription in primary glial cells. Two-directional (forward and backward) suppression subtraction hybridization (SSH) was performed on astrocytes primarily cultured from rat cerebral tissues in either standard media or treated with DA.PCR-select differential screening was used to further verifythe differentially expressed cDNA clones, and positive clones’ weresequenced and the mRNAs were re-examined on Northern blots. Fourteensequences were identified, among which l1 were homologous to knowngenes, 3 were homologous to expressed sequence tags (ESTs). The analysisof all these identified sequences suggested that comp1ex intracellu1arsigna1ing, involving cross talks with growth factor pathway, steroidhormone pathway, and/or interferon-regulated 2-5A pathway, is induced byDA in astrocytes. The target genes of the signaling pathway were fOund tofall into fOur groups, including metabolic enzymes, stress proteins, transferproteins, and growth regulation proteins. In addition, several genes havebeen established their relationship with specific neurodegenerative diseases,showing that there is an over1ap in pathogenic mechanisms of thosediseases.Molecular cloning and functional analysis of human genes especialythose human disease genes are important biological tasks at present. DA isthe predominant catecholamine neurotransmitter in the mammalian centralnervous system, and several pathological conditions have been linked todysregulation of dopaminergic transmission. In the present study, we haveisolated a series of full-length cDNAs of DA responsive genes from huamnbrain using a homologous cloning strategy.A high-quality cDNA x phagelibrary with good representation of full-length cDNAs was constructed bySmart technique.Two runs of hybridization with SSH-PCR products of DAresonsive cDNA fragmnets (see Part one) as probe were performed, and 96positive clones were selected and sequenced, 6l candidate DA responsivegenes were isolated,of which 4 new full-length cDNAs were found andsubmitted to GeneBank.Analysis of these genes suggested that the complexmolecular cascade associated with receptor activation and DA metabolismin glial cells during dopaminergic neuronal activity and the complexIVcellular and molecular mechanism of the disorder of dopaminergictransmission.Some nove1 genes isolated laid a fOundation fOr furtherfunctional research and development of theraPy for dopaminergic diseases.Two important novel genes, a putative GTPase DRP and a LIMdomain transcription factor DATl, were selected fOr further studies basedon bioinfOrmatic ana1ysis. In this study, the rat full-length DRP a更多还原