中枢神经系统髓鞘异常与精神疾病相关性的实验研究

  本文关键词：中枢神经系统髓鞘异常与精神疾病相关性的实验研究，由笔耕文化传播整理发布。

        精神分裂症(schizophrenia)是一组严重的精神疾病，全球年发病率约为0.4-1%，好发于青壮年，治疗上尚无有效的治疗方法，给患者、社会和国家造成生理、心理及经济上的沉重负担。精神分裂症的发病机制尚不清楚，目前认为是一种遗传、环境、发育异常等多因素导致的疾病。新近的研究发现精神分裂症与髓鞘异常密切相关。DNA微阵列研究发现：精神分裂症患者中枢神经系统唯一显著下调的基因为髓鞘相关基因(MAG，Transferrin，MAL，CNP，Gelsolin与ErbB3)。采用核磁共振成影(MRI)和弥散张量成像(DTI)等影像学研究发现，精神分裂症患者出现侧脑室扩大，内侧颞叶、颞上回体积减小，白质不同程度体积减小以及基底节、胼胝体、丘脑、小脑等皮层下结构的异常。尸检结果证实精神分裂症患者中枢神经系统髓鞘发育障碍和少突胶质细胞功能异常。因此，提出精神疾病髓鞘异常假说，即少突胶质细胞发育、分化异常和脱髓鞘可能是精神分裂症的发病重要机制。假设髓鞘异常是精神分裂症重要的始发因素之一，那么髓鞘损伤易感因素将可能导致髓鞘发育异常或脱髓鞘，同时诱发精神分裂症样行为学改变。年龄是精神分裂症的敏感因素之一，同时也是脱髓鞘的敏感因素。流行病学分析证明，精神分裂症最常见于15至35岁，有50%的病人在20～30岁发病，而多发性硬化等脱髓鞘疾病也常见于年轻人。少突胶质细胞转录因子2(Olig2)是一种bHLH转录因子，其家族成员还包括Olig1和Olig3。Olig2对少突胶质细胞的发育和髓鞘的形成具有非常重要的作用。更重要的是，采用基因多态分析证实Olig2表达异常是汉族人群和欧洲人群精神分裂症的易感因素。因此，本研究选择年龄和Olig2两个精神分裂症相关因素，利用小鼠脱髓鞘和发育模型，分别探讨它们在髓鞘损伤易感性或髓鞘发育异常中的作用，及其与精神分裂症样动物行为学改变之间的相关性。期望为精神分裂症等精神疾病的髓鞘异常假说发病机制提供一定的理论基础，为该类疾病的防治提供新的预防途径及治疗靶点。本实验分为以下两个部分：第一部分：Cuprizone介导的年龄相关性脱髓鞘与精神分裂症样动物行为学改变的相关性建立Cuprizone(CPZ)介导的不同年龄小鼠急性脱髓鞘动物模型，首先通过体重测量及快蓝染色鉴定模型建立是否成功；然后通过行为学检测观察脱髓鞘是否会导致精神分裂症样动物行为学改变；最后通过免疫组织化学染色及Western blot等方法观察不同年龄小鼠脑内胼胝体区及皮层区髓鞘脱失、成熟少突胶质细胞损伤及星形胶质细胞活化情况，探讨年龄与髓鞘脱失敏感性之间的内在联系，，以及成熟少突胶质细胞及星形胶质细胞于脱髓鞘过程中可能发挥的作用。主要结果如下：1、成功建立CPZ诱导的急性脱髓鞘动物模型，通过体重测量及快蓝染色证实模型小鼠均出现一定程度的体重下降及脱髓鞘。2、开场实验中，同年龄的实验组(CPZ)小鼠及对照组(CTL)小鼠在整个区域的活动路程无显著差异(p>0.05)；在中心区域，相比于CTL小鼠，各年龄组内CPZ小鼠活动路程均明显减少(p<0.05)，其中小鼠年龄越小差异越明显。3、相对于中老年小鼠，青壮年小鼠经CPZ处理后，胼胝体及皮层区MBP表达显著下降(p<0.01)。4、经CPZ处理后，各年龄CPZ小鼠胼胝体及皮层区CC1阳性细胞数均有所减少，但青壮年小鼠更加明显(p<0.01)。5、GFAP阳性细胞数于胼胝体及皮层区表现为不同程度的活化，其中青壮年小鼠较中老年小鼠更加明显(p<0.01)。以上结果表明：经CPZ诱导的不同年龄的脱髓鞘动物模型中，青壮年小鼠较中老年小鼠对于髓鞘脱失更加敏感，即青壮年小鼠更容易发生脱髓鞘，年龄是影响脱髓鞘敏感性的重要因素；行为学实验中，同样为青壮年小鼠精神行为异常更加明显；同时在髓鞘脱失过程中伴有年龄相关性的成熟少突胶质细胞的损伤及星形胶质细胞的活化，因此不同年龄成熟少突胶质细胞对于外界刺激的敏感性不同及星形胶质细胞的活化可能是导致以上结论的内在原因。第二部分：转录因子Olig2敲除导致的髓鞘发育异常与精神分裂症样动物行为学改变的相关性本研究首先利用条件性敲除少突胶质细胞内Olig2的转基因小鼠观察转录因子Olig2对髓鞘发育的影响；然后通过行为学实验(开场、高架十字迷宫)检测Olig2基因敲除小鼠的行为学改变；最后利用Olig2基因敲除小鼠建立CPZ诱导的急性脱髓鞘动物模型，观察敲除Olig2对于髓鞘损伤的影响。主要结果如下：1、正常发育过程中，敲除Olig2影响脑内髓鞘的形成。2、敲除Olig2影响少突胶质细胞的分化，但对细胞增殖没有影响。3、行为学实验中，Olig2基因敲除小鼠表现出更高的探索及好奇欲望等精神行为异常表现。4、Olig2基因敲除小鼠对CPZ诱导的脱髓鞘更加敏感。以上结果说明，敲除转录因子Olig2可以影响少突胶质细胞的分化，从而影响髓鞘的发育，但对细胞的增殖无影响；敲除Olig2小鼠会出现精神分裂症样行为异常改变；同时会影响髓鞘对于损伤刺激的敏感性。因此，转录因子Olig2异常进而导致髓鞘发育障碍可能与精神分裂症等精神疾病的发生密切相关。

    Schizophrenia is a severe psychiatric disorder with an annual incidence rate ofapproximate0.4-1%worldwide. It is a kind of disease which affects the young adults mostoften. There is no satisfactory therapy for schizophrenia as yet, which will inevitablyimpose a great burden on schizophrenic patients, the society and the countries,physiologically, psychologically and economically. The pathogenesis of schizophreniaremains unknown. It is generally accepted for the present that schizophrenia is a diseaseattributable to many factors including genes, environment, development, etc. Recent studieshave shown that schizophrenia is closely associated with myelin abnormalities. A DNAmicroarray study has examined the gene expression levels in the brain of patients withschizophrenia and the results demonstrated that a group of myelin-related genes (MAG,Transferrin, MAL, CNP, Gelsolin and ErbB3) were significantly downregulated. Imagingstudies by magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) reveallateral ventriculomegaly, atrophic temporal lobe and superior temporal gyrus, white matterdeficit, and abnormalities in basal ganglia, corpus callosum, thalamus, cerebellum and othersubcortex structures in the brain of patients with schizophrenia. Postmortem examination ofschizophrenic patients confirms myelin maldevelopment and oligodendrocyte (OL)dysfunction in their central nervous system. These findings imply a causal relationshipbetween myelin abnormalities and onset of schizophrenia. Provided that myelinabnormalities are among the primary contributing factors for schizophrenia, it is reasonableto speculate that factors sensitive to myelin injury, which can account for myelinmaldevelopment or demyelination, may well induce schizophrenia-like behavioral changes.Age is an important factor that has to be considered in schizophrenia as well as indemyelination. Epidemiological analysis demonstrates that the usual onset of schizophreniais between15and35years old, with50%of schizophrenic patients first developing thesymptoms in their twenties. Other demyelinating diseases such as multiple sclerosis are alsofrequently seen among young people. Olig2is one of the bHLH transcription factor Olig family members which plays a crucial role in OL development and myelination. Recently,genetic polymorphism analysis has proved that abnormal expression of Olig2is readilyinvolved in schizophrenia among patients from Han ethnic group and European countries.In the present study, we employed a demyelination and a development mice model toevaluate the contributions of age or Olig2to myelin injury susceptibility or myelinmaldevelopment that correlate with schizophrenia-like behaviors. Our results would providemore evidence in support of the aforementioned hypothesis that myelin abnormalities havebeen involved in the pathogenesis of schizophrenia, and thus it is of great significance toprovide new therapeutic strategies for schizophrenia and other releated disorders.The study is composed of two parts:Part1: Correlation between age-related and cuprizone-induced demyelinationand schizophrenia-like behavioral changesWe established a cuprizone-induced acute demyelination model using mice withdifferent ages. Body weight measurement and LFB staining were conducted to determinewhether the model had been successfully established; behavioral tests were then performedto observe whether demyelination would induce schizophrenia-like behaviors; finallyimmunohistostaining and Western blot analysis were applied to observe levels ofdemyelination, mature OL damage and astrocytosis in corpus callosum and cortex of mousebrain. The potential correlation between age and demyelination sensitivity, as well as thecontributions of mature oligodendrocytes and astrocytes in the process of demyelination,have been investigated based on the outcomes.The results were as follows:1. The cuprizone-induced acute demyelination model was successfully established;body weight measurement and LFB staining confirmed body weight loss and demyelinationin model mice.2. In the open field test, the locomotive distances in the whole field were comparablebetween model mice and control mice of the same age (p>0.05); the locomotive distances ofmodel mice in the central area were significantly shorter than that of control mice in eachage group (p<0.05), and this decrease in distance was more significant in the younger agegroup.3. A significant downregulation of MBP was detected in corpus callosum and cortex areas of young adult mice treated with cuprizone when compared with adult and aged mice(p<0.01).4. A decrease in the number of CC1positive OL cells was observed in corpus callosumand cortex areas of model mice treated with cuprizone in each age group, and the decreasewas more significant in young adult mice than in adult and aged mice (p<0.01).5. Proliferation of GFAP positive astrocytes (ASTs) was observed in corpus callosumand cortex areas of CPZ mice, and the level of proliferation was more significant in youngadult mice than in adult and aged mice (p<0.01).Based on the above experimental results, we can reach the following conclusions:young adult mice are more sensitive to demyelination compared with adult and aged mice inthat they show higher proneness to demyelination; behavioral tests demonstrate thatabnormalities of psychiatric behaviors are more evident in young adult mice; the process ofdemyelination is accompanied with age-related damage of mature oligodendrocytes andastrocytosis. Therefore, age-dependent sensitivity of mature oligodendrocytes todemyelination and astrocytosis may be the underlying causes that can account for the aboveconclusions.Part2: Correlation between Olig2knockout myelin development andschizophrenia-like behavioral changesIn this part, the function of transcription factor Olig2in myelin development wasobserved by knocking out Olig2in OLs using CNP promoter; behavioral tests (open fieldand elevated plus-maze tests) were then performed to evaluate behavioral changes in Olig2knockout mice; and finally a cuprizone-induced acute demyelination model was establishedusing the Olig2knockout mice to observe the influence of Olig2on myelin damage.The results were as follows:1. Knockout of Olig2in OLs obstructed myelination in nomal brain development.2. Knockout of Olig2inhibited differentiation of OLs, but showed little influence oncell proliferation.3. Behavioral tests confirmed abnormalities of psychiatric behaviors in Olig2knockoutmice in that they showed stronger desire for exploration and stronger curiosity whencompared with control mice.4. Olig2knockout mice were more sensitive to cuprizone-induced demyelination. Based on the above experimental results, we can reach the following conclusions:knockout of transcription factor Olig2can affect oligodendrocyte differentiation andthereby affect myelin development; Olig2knockout mice tend to conduct abnormalpsychiatric behaviors and exhibit more sensitivity to myelin damage. Taking together, themyelin maldevelopment resulted by abnormalities of transcription factor Olig2may closelyassociated with the onset of schizophrenia and other psychiatric disorders.

        中枢神经系统髓鞘异常与精神疾病相关性的实验研究

英文缩写一览表4-5英文摘要5-8中文摘要9-12前言12-16    参考文献14-16总材料和方法16-27第一部分 Cuprizone介导的年龄相关性脱髓鞘与精神分裂症样动物行为学改变的相关性27-44    材料和方法28-30    结果30-37    讨论37-39    小结39-40    参考文献40-44第二部分 转录因子Olig2敲除导致的髓鞘发育异常与精神分裂症样动物行为学改变的相关性44-60    材料和方法45-48    结果48-55    讨论55-57    小结57-58    参考文献58-60全文结论60-61致谢61-62文献综述 少突胶质细胞发育与细胞易损性研究进展62-69    参考文献66-69攻读硕士期间论文完成情况及参加的重要学术活动69

  本文关键词：中枢神经系统髓鞘异常与精神疾病相关性的实验研究，由笔耕文化传播整理发布。