【作者】 王晗知；

【导师】 肖岚； 蔡文琴；

【作者基本信息】 第三军医大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 精神分裂症是一组病因未明的重性精神病,全球年发病率为0.4-1%,给患者、社会和国家造成生理上、经济上及社会和谐的沉重负担。近年来在对精神分裂症的研究中发现精神分裂症患者均有不同程度的脑白质异常以及少突胶质细胞谱系(oligodendrocyte lineage cells, OLs)和髓鞘病理改变。DNA微阵列分析发现精神分裂症患者的多个脑区皮层髓鞘相关基因,包括CNP、MAG、GALC、MOG等表达明显下调。影像学更进一步证实精神分裂症患者MRI核磁共振显影观察到侧脑室扩大,内侧颞叶,颞上回皮层、顶叶体积减少,全脑或局部白质体积有不同程度的减少,胼胝体、小脑、丘脑等皮层下结构异常。以上研究均提示,精神分裂症认知障碍可能起因于神经元相关髓鞘体积减少从而导致上述结构的信号转导紊乱。所以少突胶质细胞功能异常有可能是精神分裂症发病原因之一,为此少突胶质细胞不失为精神分裂症治疗的靶点。由于精神分裂症的病因未明,因此目前对精神分裂症的一线治疗主要仍依靠使用抗精神病药物治疗。典型性抗精神病药,如氟哌啶醇(Haloperidol,HAL),主要通过抑制多巴胺D2类受体(包括D2r、D3r和D4r),进行药物治疗。尽管HAL能有效改善精神分裂症的阳性症状,却无法有效治疗阴性症状,也无法逆转病程。近年来,许多非典型性抗精神病药,如喹硫平(Quetiapine, QUE)、奥氮平(Olanzopine, OLA)等,通过双向调节多巴胺、5羟色胺等神经递质受体,能显著改善精神分裂症的阴性症状,部分能改善阳性症状,且能有效的逆转病情的进程。最近系列研究提示,作为白质主要细胞成分的少突胶质细胞与抗精神病药的药物作用存在潜在的关联,但抗精神病药物对少突胶质细胞发育或髓鞘修复的作用事实上并不明确。本研究拟以抗精神病药为工具,比较典型性及非典型性抗精神病药对少突胶质细胞发育的作用,进而利用中枢神经脱髓鞘模型,比较两类抗精神病药对脱髓鞘的再生修复以及对动物行为学变化的影响,并检测非典型性抗精神病药可能促进少突胶质细胞发生的细胞学机制。为少突胶质细胞的退变参与精神分裂症病理生理过程的阐明提供神经药理学实验依据,并为中枢神经脱髓鞘疾病治疗的药物开发研究提供新的线索。本实验分为以下三个部分:第一部分:抗精神病药对成年小鼠大脑少突胶质细胞前体细胞(Oligododendrocyte precursor cells,OPCs)的影响通过慢性摄入两类抗精神病药HAL、QUE,分别检测抗精神病药对大脑中OLs细胞的发育的影响,阐明不同脑区的OLs分布、密度的变化。主要结果如下:1. HAL可以抑制小鼠在开场试验中的自发活动的活性和对陌生环境的探索行为。2. HAL可以促进成年小鼠脑胼胝体NG2+细胞增生。3. HAL可以促进成年小鼠大脑各脑区(胼胝体、海马、皮层)Olig2+OPCs增生以及Olig2蛋白表达量增加。4. HAL对APC+、GFAP+、CD68+细胞数目没有改变。5. QUE对正常小鼠大脑中OLs无任何影响。以上结果表明HAL可以促进成年小鼠脑内OPCs增生且不引起神经胶质细胞增生,并从另一个方面提示OPCs和精神分裂症病理机制的关系,即OPCs可能是抗精神病药作用的靶点。第二部分:抗精神病药对脱髓鞘模型中髓鞘修复的影响本研究通过建立Cuprizone诱导的小鼠脱髓鞘模型,分别观察两类抗精神病药HAL、QUE对髓鞘修复的作用,明确脑白质损伤在精神分裂症病理机制中的作用,同时了解脱髓鞘疾病的髓鞘再生机制。主要结果如下:1.成功建立Cuprizone诱导的脱髓鞘动物模型。0.2% Cupriozone摄入6周,胼胝体脱髓鞘损伤达到最大,停药3周后,髓鞘自发性修复,几乎达到正常水平。持续摄入0.2% cuprizone12周后停药3周,仍可见较低水平的髓鞘修复。2. HAL可以推迟髓鞘的自发性修复。QUE能促进慢性髓鞘再生的进程。3. HAL在急性髓鞘修复过程中可以促进OPCs增生活化,但是在慢性髓鞘修复中HAL使OPCs数目减少。QUE可以促进慢性髓鞘修复中的OPCs增生。4. QUE可以减少髓鞘修复过程中小胶质细胞的募集和活化。以上结果说明,两类抗精神病药物对髓鞘的修复作用有所不同:HAL抑制髓鞘的修复再生,在急性髓鞘修复中促进OPCs增生,但在慢性髓鞘修复过程中减少OPCs细胞数量;而QUE在慢性髓鞘修复中诱导OPCs数量的增加,促进髓鞘再生。此外,QUE抑制小胶质细胞在髓鞘损伤部位的活化和聚集。第三部分:喹硫平调节脂多糖诱导的小胶质细胞活化的实验研究本研究通过建立脂多糖(LPS)诱导的小胶质N9细胞活化模型,观察QUE对小胶质细胞活化的影响,结合细胞培养、流式细胞术、免疫荧光细胞化学等,探讨QUE对小胶质细胞活化的影响极其调控方式。主要结果如下:1.建立LPS诱导的N9小胶质细胞活化模型,证明LPS并不影响N9细胞增殖,但促进NO释放而使细胞活化。2. QUE(10nM)可以减少LPS激活N9细胞所释放的NO产物含量,以及减少CD11b在胞膜的表达量。从而抑制N9细胞的活化。3. QUE能抑制NFκB的活化及位移入核。以上结果说明, QUE可以抑制LPS诱导的N9小胶质细胞的活化,并且这种作用可能通过抑制小胶质细胞中NFκB的活化来达成。综上所述:在正常小鼠体内,HAL可以激活成年小鼠大脑内静息的OPCs细胞;QUE对正常小鼠没有明显作用。在脱髓鞘模型中,HAL抑制髓鞘自发修复,QUE可以促进慢性髓鞘修复的进程,其机制可能与两种药物对OPCs的不同作用有关。因此,OPCs可能是抗精神病药治疗的靶点。此外,QUE还能抑制脱髓鞘损伤部位的小胶质细胞的活化,可能也是QUE促进髓鞘修复的原因之一。更多还原

【Abstract】 Schizophrenia is one kind of devastating mental disorder of unknown etiology that affects approximately 0.4-0.6% of general population per year. The impacts may include the psychological, social and financial resources of the patient, family and large community.Recent study results showed that the abnormality of white matter and pathological changes of oligodendrocyte lineage cells and myelination existed in several brain areas of schizophrenia. DNA microarray analyses have been performed in post-mortem brain regions of patients and healthy individuals to investigate the down-regulation of myelin-related genes, including CNP, MAG, GALC, and MOG. Further evidence for the role of oligodendrocytes and myelin in schizophrenia is obtained from imaging studies. Magnetic resonance imaging (MRI) findings in patients diagnosed with schizophrenia include lateral ventricle enlargement, medial temporal lobe volume reduction, neocortical superior temporal gyrus volume reduction, parietal lobe abnormalities, global and regional reduction in white matter volume, such as corpus callosum, and subcortical abnormalities affecting the basal ganglia, thalamus and cerebellum. These findings reveal congnitive dysfunction of schizophrenia possibly result from disconnection in both local neural circuitries that spanning several brain areas. It is assumed that the oligodendrocyte may be one of the therapeutic targets in the treatment of the patients with of schizophrenia.Based on the limited understanding on etiology of schizophrenia, the first line psychiatric treatment for schizophrenia is antipsychotic medication. The typical antipsychotic drugs, such as haloperidol, are dopamine D2-like subfamily receptor (D2r, D3r and D4r) antagonists. Although typical antipsychotic drugs can reduce the positive symptoms of psychosis, they fail neither to significantly ameliorate the negative symptoms, nor reverse the progress of mental disorder. Currently available atypical antipsychotic drugs, such as quetiapine, clozapine, olanzapine, can improve both positive and negative symptoms. These drugs can therefore prevent aggravation of psychotic symptoms, by modulating multiple neurotransmitter receptors, including dopamine receptor, 5-HT receptor.Recently, a series of studies suggest that a potential relationship between oligodendroglia, the main component of white matter regions of the brain and schizophrenia. aIt is, however, still not clear about the effect of antipsychotics treatmen on oligodendrocyte/myelin in brain.In this study, we have investigated the effect of antipsychotic on oligodendrocyte linage in brain of mice, and the remyelination in cuprizone-induced demyelin model, by using two kind of antipsychotic in comparison. The results presented in this paper may provide us a new insight into the degeneration of oligodendrocyte, which may involve in the pathophysiology of schizonphrenia, and reveal a new clue to pharmaprojects for demyelianaton disease in central neural system.This investigation is composed of three parts:Part 1: The effect of antipsychotic on OPCs in brain of adult miceYoung adult C57Bl/6 mice were given antipsychotic treatment in their drinking water for three or six weeks. Based on the investigations of the effect of two kind of antipsychotic on the development of oligodendrocyte lineage, we found that:1. HAL decreases the locomotion of animal in open-field test.2. HAL increases the number of NG2-expression cells in the corpus callosum.3. haloperidol increases the number of Olig2-expressiong cells and the amount of Olig2 protein4. haloperidol treatment has no effect on the maturation of OLs, and also has no effect on the numbers of GFAP+ and CD68+ cells in the brain.5. QUE has no effect on the oligodendrocyte lineage in brain of healthy adult mice.The first part revealed that chronic haloperidol treatment activates quiescent OPCs in adult mouse brain in the absence of gliosis. OPCs are potential targets of antipsychotic medication.Part 2: The effect of antipsychotic on the remyelination of cuprizone-induced demyelin model.To establish the cuprizone-induced demyelination animal model and to investigate the effect of two kind of antipsychotic haloperidol, quetiapine on remyelination, the studies plan to clarify the role of dysfunction of white matter in pathophysiology of schizonphrenia, and to investigate the molecular mechinsm of remyelination in demyelin diseases. The findings are as follows:1. The establishment cuprizone-induced demyelination model, using a diet supplemented with 0.2% cuprizone. The loss of myelin is more pronounced in mice fed the cuprizone diet at 6 weeks. There is spontaneous remyeliantion fowllowing the subsequent 3 weeks when mice are fed normal chow. For the prolonged administration of cuprizone for 12 weeks, the extent of remyelination turns to be very limited when mice change to normal chow.2. Haloperidol can delay the progress of spontaneous remyeliantion; quetiapine can promote the progress of spontaneous remyeliantion in chronic demyelination model.3. In acute demyeliantion model, haloperidol can increase the number of Olig2-expression cells, but decrease the number of this type of cells in chronic demyelination model. Quetiapine can increase the the number of Olig2-expression cells in chronic demyelination model.4. Quetiapine can reduce the recruitment and activation of microglia/macrophage in lesion location of demyeliantion.The second part revealed that we established the cuprizone-induced demyeliantion animal model successfully. Different kinds of antipsychotic play different roles in remyelination: Haloperidol can inhibit the remyelination while quetiapine can promote the progress of remyelination. Haloperidol can increase the number of oligodendrocyge precursor cells (OPCs) in acute demyelination, but decrease this type of cell in chronic demyelination. Quetiapine can increase the OPCs in chronic demyeliantion progress. Additionally, quetiapine can inhibit the recruitment and activation of microglia/macrophage in lesion location of demyeliantion.Part 3: Quetiapine can reduce LPS-induced activation of microglia.This study use lipopolysaccharide (LPS)-activated mouse microglial cell line N9 as an in vitro model to mimic microglia activation seen in mouse. The effects of quetiapine on the LPS-stimulate N9 cells were investigated. The findings are as followings:1. Cell culture and identification of N9 cell. To choose the antibody CD11b to identification N9 cells and count the positive cells, more than 95% N9 cell can be marked with CD11b.2. The establishement of LPS-activated N9 cells model. LPS do not affect the cell viability of N9 cell. However, the release of NO by LPS-stimulated N9 cells resulted in significant increase, which proves the successful establishment of LPS-activated N9 cells.3. Quetiapine can reduce the activation of LPS-stimulated N9 cells. Quetiapine can decrease the NO production induced by LPS-stimulated N9 cells, and decrease the expression amount of CD11b in cell membrane.4. Quetiapine can inhibit the activation of NFκB and prevent the immigration of NFκB to nuclear.The third part revealed that we established the LPS-activated N9 cells model successfully. Quetiapine can inhibit the activation of LPS-stimulated N9 cells. Quetiapine may regulat the activation of NFκB to inhibit the activation of N9 cells.In summary, this study demonstrated that chronic haloperidol treatment activates quiescent OPCs in the brain of normal adult mouse. Haloperidol can delay the progress of spontaneous remyeliantion while quetiapine can promote the progress of spontaneous remyeliantion in chronic demyelination model. The mechanism may be due to the different effects of these two kinds of antipsychotic on OPCs. Therefore, OPCs is a potential target of antipsychotic medication. Additionally, quetiapine can inhibit the recruitments and activation of microglia/macrophage in lesion location of demyeliantion through regulating the activation of NFκB. This may be one of the mechanisms underlying the effect of quetiapine in promotion of remyelination.更多还原