本文關(guān)鍵詞：中樞神經(jīng)系統(tǒng)髓鞘異常與精神疾病相關(guān)性的實驗研究，由筆耕文化傳播整理發(fā)布。

        精神分裂癥(schizophrenia)是一組嚴(yán)重的精神疾病，全球年發(fā)病率約為0.4-1%，好發(fā)于青壯年，治療上尚無有效的治療方法，給患者、社會和國家造成生理、心理及經(jīng)濟上的沉重負擔(dān)。精神分裂癥的發(fā)病機制尚不清楚，目前認為是一種遺傳、環(huán)境、發(fā)育異常等多因素導(dǎo)致的疾病。新近的研究發(fā)現(xiàn)精神分裂癥與髓鞘異常密切相關(guān)。DNA微陣列研究發(fā)現(xiàn)：精神分裂癥患者中樞神經(jīng)系統(tǒng)唯一顯著下調(diào)的基因為髓鞘相關(guān)基因(MAG，Transferrin，MAL，CNP，Gelsolin與ErbB3)。采用核磁共振成影(MRI)和彌散張量成像(DTI)等影像學(xué)研究發(fā)現(xiàn)，精神分裂癥患者出現(xiàn)側(cè)腦室擴大，內(nèi)側(cè)顳葉、顳上回體積減小，白質(zhì)不同程度體積減小以及基底節(jié)、胼胝體、丘腦、小腦等皮層下結(jié)構(gòu)的異常。尸檢結(jié)果證實精神分裂癥患者中樞神經(jīng)系統(tǒng)髓鞘發(fā)育障礙和少突膠質(zhì)細胞功能異常。因此，提出精神疾病髓鞘異常假說，即少突膠質(zhì)細胞發(fā)育、分化異常和脫髓鞘可能是精神分裂癥的發(fā)病重要機制。假設(shè)髓鞘異常是精神分裂癥重要的始發(fā)因素之一，那么髓鞘損傷易感因素將可能導(dǎo)致髓鞘發(fā)育異�；蛎撍枨剩瑫r誘發(fā)精神分裂癥樣行為學(xué)改變。年齡是精神分裂癥的敏感因素之一，同時也是脫髓鞘的敏感因素。流行病學(xué)分析證明，精神分裂癥最常見于15至35歲，有50%的病人在20～30歲發(fā)病，而多發(fā)性硬化等脫髓鞘疾病也常見于年輕人。少突膠質(zhì)細胞轉(zhuǎn)錄因子2(Olig2)是一種bHLH轉(zhuǎn)錄因子，其家族成員還包括Olig1和Olig3。Olig2對少突膠質(zhì)細胞的發(fā)育和髓鞘的形成具有非常重要的作用。更重要的是，采用基因多態(tài)分析證實Olig2表達異常是漢族人群和歐洲人群精神分裂癥的易感因素。因此，本研究選擇年齡和Olig2兩個精神分裂癥相關(guān)因素，利用小鼠脫髓鞘和發(fā)育模型，分別探討它們在髓鞘損傷易感性或髓鞘發(fā)育異常中的作用，及其與精神分裂癥樣動物行為學(xué)改變之間的相關(guān)性。期望為精神分裂癥等精神疾病的髓鞘異常假說發(fā)病機制提供一定的理論基礎(chǔ)，為該類疾病的防治提供新的預(yù)防途徑及治療靶點。本實驗分為以下兩個部分：第一部分：Cuprizone介導(dǎo)的年齡相關(guān)性脫髓鞘與精神分裂癥樣動物行為學(xué)改變的相關(guān)性建立Cuprizone(CPZ)介導(dǎo)的不同年齡小鼠急性脫髓鞘動物模型，首先通過體重測量及快藍染色鑒定模型建立是否成功；然后通過行為學(xué)檢測觀察脫髓鞘是否會導(dǎo)致精神分裂癥樣動物行為學(xué)改變；最后通過免疫組織化學(xué)染色及Western blot等方法觀察不同年齡小鼠腦內(nèi)胼胝體區(qū)及皮層區(qū)髓鞘脫失、成熟少突膠質(zhì)細胞損傷及星形膠質(zhì)細胞活化情況，探討年齡與髓鞘脫失敏感性之間的內(nèi)在聯(lián)系，，以及成熟少突膠質(zhì)細胞及星形膠質(zhì)細胞于脫髓鞘過程中可能發(fā)揮的作用。主要結(jié)果如下：1、成功建立CPZ誘導(dǎo)的急性脫髓鞘動物模型，通過體重測量及快藍染色證實模型小鼠均出現(xiàn)一定程度的體重下降及脫髓鞘。2、開場實驗中，同年齡的實驗組(CPZ)小鼠及對照組(CTL)小鼠在整個區(qū)域的活動路程無顯著差異(p>0.05)；在中心區(qū)域，相比于CTL小鼠，各年齡組內(nèi)CPZ小鼠活動路程均明顯減少(p<0.05)，其中小鼠年齡越小差異越明顯。3、相對于中老年小鼠，青壯年小鼠經(jīng)CPZ處理后，胼胝體及皮層區(qū)MBP表達顯著下降(p<0.01)。4、經(jīng)CPZ處理后，各年齡CPZ小鼠胼胝體及皮層區(qū)CC1陽性細胞數(shù)均有所減少，但青壯年小鼠更加明顯(p<0.01)。5、GFAP陽性細胞數(shù)于胼胝體及皮層區(qū)表現(xiàn)為不同程度的活化，其中青壯年小鼠較中老年小鼠更加明顯(p<0.01)。以上結(jié)果表明：經(jīng)CPZ誘導(dǎo)的不同年齡的脫髓鞘動物模型中，青壯年小鼠較中老年小鼠對于髓鞘脫失更加敏感，即青壯年小鼠更容易發(fā)生脫髓鞘，年齡是影響脫髓鞘敏感性的重要因素；行為學(xué)實驗中，同樣為青壯年小鼠精神行為異常更加明顯；同時在髓鞘脫失過程中伴有年齡相關(guān)性的成熟少突膠質(zhì)細胞的損傷及星形膠質(zhì)細胞的活化，因此不同年齡成熟少突膠質(zhì)細胞對于外界刺激的敏感性不同及星形膠質(zhì)細胞的活化可能是導(dǎo)致以上結(jié)論的內(nèi)在原因。第二部分：轉(zhuǎn)錄因子Olig2敲除導(dǎo)致的髓鞘發(fā)育異常與精神分裂癥樣動物行為學(xué)改變的相關(guān)性本研究首先利用條件性敲除少突膠質(zhì)細胞內(nèi)Olig2的轉(zhuǎn)基因小鼠觀察轉(zhuǎn)錄因子Olig2對髓鞘發(fā)育的影響；然后通過行為學(xué)實驗(開場、高架十字迷宮)檢測Olig2基因敲除小鼠的行為學(xué)改變；最后利用Olig2基因敲除小鼠建立CPZ誘導(dǎo)的急性脫髓鞘動物模型，觀察敲除Olig2對于髓鞘損傷的影響。主要結(jié)果如下：1、正常發(fā)育過程中，敲除Olig2影響腦內(nèi)髓鞘的形成。2、敲除Olig2影響少突膠質(zhì)細胞的分化，但對細胞增殖沒有影響。3、行為學(xué)實驗中，Olig2基因敲除小鼠表現(xiàn)出更高的探索及好奇欲望等精神行為異常表現(xiàn)。4、Olig2基因敲除小鼠對CPZ誘導(dǎo)的脫髓鞘更加敏感。以上結(jié)果說明，敲除轉(zhuǎn)錄因子Olig2可以影響少突膠質(zhì)細胞的分化，從而影響髓鞘的發(fā)育，但對細胞的增殖無影響；敲除Olig2小鼠會出現(xiàn)精神分裂癥樣行為異常改變；同時會影響髓鞘對于損傷刺激的敏感性。因此，轉(zhuǎn)錄因子Olig2異常進而導(dǎo)致髓鞘發(fā)育障礙可能與精神分裂癥等精神疾病的發(fā)生密切相關(guān)。

    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.

        中樞神經(jīng)系統(tǒng)髓鞘異常與精神疾病相關(guān)性的實驗研究

英文縮寫一覽表4-5英文摘要5-8中文摘要9-12前言12-16    參考文獻14-16總材料和方法16-27第一部分 Cuprizone介導(dǎo)的年齡相關(guān)性脫髓鞘與精神分裂癥樣動物行為學(xué)改變的相關(guān)性27-44    材料和方法28-30    結(jié)果30-37    討論37-39    小結(jié)39-40    參考文獻40-44第二部分 轉(zhuǎn)錄因子Olig2敲除導(dǎo)致的髓鞘發(fā)育異常與精神分裂癥樣動物行為學(xué)改變的相關(guān)性44-60    材料和方法45-48    結(jié)果48-55    討論55-57    小結(jié)57-58    參考文獻58-60全文結(jié)論60-61致謝61-62文獻綜述 少突膠質(zhì)細胞發(fā)育與細胞易損性研究進展62-69    參考文獻66-69攻讀碩士期間論文完成情況及參加的重要學(xué)術(shù)活動69

  本文關(guān)鍵詞：中樞神經(jīng)系統(tǒng)髓鞘異常與精神疾病相關(guān)性的實驗研究，由筆耕文化傳播整理發(fā)布。