【作者】 魏秀岩；

【导师】 吴春福；

【作者基本信息】 沈阳药科大学 ， 药理学， 2007， 博士

【摘要】 随着社会的发展，精神障碍越来越受到人们的重视，在10种造成社会最沉重负担的疾病中，精神疾病占了4种。精神医学正在逐步受到医学同行及社会的关注并被赋予新的认识。虽然各国学者对焦虑症和抑郁症等精神障碍展开了大规模的研究工作，积累了大量的资料，但到目前为止，对焦虑症和抑郁症等精神障碍的发病机制仍是众说纷纭，尚未有统一的看法。动物实验是研究焦虑症和抑郁症病理生理学以及抗焦虑和抗抑郁药物筛选的重要方法之一。大量研究已发现孤养模型动物有神经、内分泌、免疫等多个系统的结构和功能的病理变化，长期孤养可引起脑的发育异常并引起持久的精神障碍，包括焦虑、抑郁、攻击等多种行为变化，可以模拟人类的精神障碍。本实验应用小鼠孤养模型，用一系列焦虑和抑郁动物模型研究了孤养小鼠的行为学变化，并考察了油酰胺、黄芩苷、西洋参皂苷几种药物对其行为学变化的影响。结果发现，与群养小鼠比较，孤养模型小鼠在高架十字迷宫、明暗箱、孔板试验、隔离诱导的攻击实验中表现为焦虑和攻击行为，在强迫游泳和悬尾实验中表现为抑郁行为。几种药物均可不同程度上改善孤养小鼠的焦虑状态，证实它们对孤养小鼠具有抗焦虑作用。此外，油酰胺和黄芩苷对孤养小鼠还有抗抑郁作用。由于油酰胺是近年发现的内源性生物活性分子，具有多种药理学作用，各国学者对其作用和机制展开了研究，但其作用机理复杂，至今尚未阐明。本研究采用免疫组化和免疫印迹的方法考察了油酰胺对孤养小鼠海马和前额叶皮层两个脑区的5-HT_1A、5-HT_2A和GABA_A受体的影响。结果发现，孤养小鼠5-HT_1A和5-HT_2A受体表达上调，GABA_A受体下调，油酰胺可以逆转这些变化。提示油酰胺可能作用于5-HT能和GABA_A能受体而发挥作用。蛋白质组学可以比较两个或更多的样品之间蛋白质整体表达水平，是揭示疾病分子机制，发现疾病标志蛋白，寻找新的药物靶标的理想工具，也是生命科学进入后基因组时代的标志之一。本实验利用二维电泳结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）技术进一步考察油酰胺对孤养小鼠海马和前额叶皮层蛋白质组的影响。本实验比较了群养对照组、孤养模型组和油酰胺给药组小鼠海马和前额叶皮层组织的双向电泳图谱，采用Melanie 4.0双向电泳图谱分析软件获得了三组之间的差异蛋白质。通过MALDI-TOF-MS质谱分析，鉴定出了部分与孤养模型相关的蛋白质及油酰胺发挥对孤养小鼠抗焦虑和抗抑郁作用的部分靶蛋白。根据这些蛋白质的功能，可将它们分为如下几类：细胞骨架及其相关蛋白[包括微管蛋白（tubulin），神经丝蛋白（NF-M）和原肌球蛋白（TPM3）]、酶类[包括ATP合酶（ATP synthase），烯醇化酶（enolase），异柠檬酸脱氢酶（IDH3A），泛醇细胞色素c还原酶（UQCR1）]，热休克蛋白47（HSP47），信号转导蛋白[如GDP解离抑制因子（GDI）]等。为进一步研究孤养模型发生的分子机制及油酰胺治疗作用奠定了基础。总之，本实验从药理学角度考察几种药物特别是油酰胺对孤养模型小鼠的行为学变化、受体水平的变化以及蛋白质组学的改变，为进一步研究焦虑症和抑郁症发生机制及用此模型研究药物的相关作用提供了科学依据。更多还原

【Abstract】 Animal models are important tools for the advancement of medicine, and have increasing importance in psychiatric studies. Social isolation of rodents is used to mimic human psychopathological processes, such as depression and anxiety. Investigation of the pattern of behavioral changes in mice reared in isolation may help our understanding of the aetiology of human anxiety and depression disorders.The present studies demonstrated that social-isolated mice produced the anxiogenic and aggressive profile by using the elevated plus-maze test, the light/dark test, the hole-board test. Treatment with several drugs, including oleamide, baicalin and Panaxquin quefoliuml saponin （PQS） produced the anxiolytic and anti-aggressive effects in social-isolated mice. In addition, the result also showed that oleamide and baicalin had an antidepressant-like effect in social-isolated mice by using two depressive models in mice, i.e. forced swimming test （FST） and tail suspension test （TST）.Oleamide （cis-9,10-octadecenoamide） is an endogenous sleep-inducing substance, first isolated from the cerebrospinal fluid of sleep-deprived cats. In recent years, accumulating evidence supports that oleamide is an endogenous signaling factor and has many pharmacological activities. But the mechanism of action of oleamide in the central nervous system remains unclear.To further explore the mechanisms of action underlying the anxiolytic and anti-depressant activities of oleamide, 5-HT_1A, 5-HT_2A and GABA_a receptor expression were determined in the hippocampal and prefrontal cortex of the mice by using western blot analysis and immunohistochemical technique. The results showed that expression of 5-HT_1A and 5-HT_2A receptors was increased and expression of GABA_A receptor was decreased in social-isolated mice, which could be reversed by oleamide. Thus, we suggested that the anxiolytic and anti-depressive effect of oleamide may be related with serotonergic and GABAergic receptors.Proteomic technique is now obviously the overwhelming used method in studies of proteins differentially expressed in altered conditions. In the present study, proteomic analysis by two-dimensional gel electrophoresis （2-DE） together with mass spectrometry （MS） was applied to compare hippocampus and prefrontal cortex protein profiles among different groups [group-housed mice（GH）、social-isolated mice（SI）、SI+oleamide20mg/kg]. After differential analysis and identification by MALDI-TOF/MS, structural proteins [tubulin, neurofilaments protein M （NF-M） and tropomyosin gamma （TPM3）], enzymes with various catalytic activities [ATP synthase, enolase, isocitrate dehydrogenase （IDH3A）, Ubiquinol-cytochrome-c reductase complex core protein I （UQCR1）], signal associated protein [guanine nucleotide dissociation inhibitor （GDI）, Protein kinase C inhibitor protein 1（14-3-3 protein zeta/delta）], heat shock protein 47 （HSP47） were found differentially expressed between hippocampus in social-isolated and group-housed mice, and some of them could be reversed by oleamide. The results of proteomics settled the foundation for further studying the molecular mechanism of social isolation and the role of oleamide.In conclusion, the report studied the effect of behavioral changes of several drugs, especially oleamide, the effect of receptors and proteomic changes of oleamide on social-isolatded mice. It offers scientific proofs for the study of mechanism of anxiety and depression and correlated effect of drugs by using the social-isolated model.更多还原