【作者】 方芳；

【导师】 刘娅； 李新民；

【作者基本信息】 吉林大学 ， 卫生毒理学， 2010， 博士

【摘要】 精神分裂症是一类常见的、慢性的可导致精神残疾的疾病,主要以思维、情感、行为等多方面的障碍及精神活动的不协调为主要特点,其患病率大约为1%。可以给个人、家庭和社会带来巨大的痛苦和负担,因此对精神分裂症的合理治疗一直是一个重要课题。目前精神分裂症的治疗主要是应用抗精神病药物,包括典型抗精神病药物和非典型抗精神病药物。少突胶质细胞功能障碍和死亡在精神分裂症等精神障碍及多发硬化等神经退行性病变的病理改变中具有重要作用。前期研究表明,上述疾病中白质和少突胶质细胞均存在一定的损伤和死亡。神经干细胞原代培养发现,抗精神病药物可明显增加少突胶质细胞的数量,并促进神经干细胞向成熟少突胶质细胞分化,但药物产生这种效果的具体机制尚不明确。此效应是否特异地作用于少突胶质细胞系,尚未阐明,本研究以大鼠少突胶质细胞系CG4细胞为研究对象,研究非典型抗精神病药物奎硫平和奥氮平以及典型抗精神病药物氟哌啶醇直接作用少突胶质前体细胞,探讨抗精神病药物对少突胶质细胞系的直接作用机制,进一步探索精神分裂症的发病机制,为更好地研发高效、副作用小的新型抗精神病药物提供理论依据和实验数据。本研究结果表明,奎硫平、奥氮平和氟哌啶醇3种抗精神病药物对CG4细胞均无明显的促增殖作用,并在较高浓度和较长时间培养条件下对细胞具有毒性效应;这3种药物均具有促进CG4细胞向成熟少突胶质细胞分化的效应,非典型抗精神病药物奎硫平和奥氮平促少突胶质细胞分化效应可能由Olig1/Olig2(bHLH)转录因子介导,而典型抗精神病药物氟哌啶醇的促分化效应可能只通过Olig2转录因子介导。更多还原

【Abstract】 In recent decades, with global socio-economic rapidly developing and pace of life expediting, people face with more pressure from family, life and work, then leading to increasing number of people suffering from insomnia, anxiety, mania, depression, schizophrenia, Parkinson’s and senile dementia and other neurological and psychiatric disorders. These kinds of diseases are mostly chronic and persistent, once onset would cause a great burden to the family and society, which has attracted extensive attention from the whole society and has become great challenges in the 21st century.At present, antipsychotic drugs (APDs) are primarily treatment of mental disorders such as schizophrenia. However, some patients are not sensitive to drug treatment or lack of compliance, which cause residual symptoms, recurrence and companied with some other diseases. Most patients with schizophrenia require long-term or even lifelong treatment and support. Schizophrenia is one of the major diseases that consume more medical resources because of its high morbidity and recurrence rates in China. Recent years, the consumption of APDs has a clear upward trend. Comparing and selecting economic, safe and effective treatment for schizophrenia and assessing the prognosis of schizophrenia, which is currently the world’s concern. The study of new atypical APDs has great significance, which not only help to solve problems of the typical APDs, but also can be used as a probe exploring human learning, work capacity and cognitive function.Recent studies found that oligodendrocyte dysfunction and death plays an important role in the pathological changes of schizophrenia. In the CNS, myelin is synthesized by oligodendrocytes. The myelin coating of axons is a prerequisite for rapid impulse conduction and maintenance of axonal function. Whereas oligodendrocyte and myelin have important function and oligodendrocytes have a high susceptibility to injuries of the CNS, understanding mechanism of oligodendrocytes apoptosis and injury and exploring proliferation of remyelination can be new therapeutic strategies.In this study, the subject is a rat oligodendrocyte cell line, CG4 cells, investigating a direct role of a typical APD haloperidol (HAL) and atypical APDs quetiapine (QUE) and olanzapine (OLA) on oligodendrocyte precursor cells, which can better explore the pathogenesis of schizophrenia and provide a theoretical basis and experimental data of studying new APDs.Methods: Using MTT、CCK-8 assay and BrdU assay to test the toxicity and proliferation effects of APDs on CG4 cells, and using optical microscope, immunofluorescence staining and Western Blot to detect differentiation effects of APDs on CG4 cells.Treatment of toxicity and proliferation effects on CG4 cells: CG4 cells were seeded into 96-well plates coated with PDL to culture for 16 h. Changing fresh medium (growth medium, GM) and adding APDs. Drug concentrations were as follows: 0, 0.01, 0.1, 1, 10 and 100μM. Placing plates into an incubator (5% CO2, 37℃), and then respectively testing 24 h, 48 h and 72 h results.Treatment of differentiation effects on CG4 cells: CG4 cells were seeded into 6-well plates or chambers coated with PDL to culture for 16 h. Changing the GM to differentiation medium (DM), and adding the different concentration of APDs. Drug concentrations were as follows: 0, 0.01, 0.05, 0.1, 0.5 and 1μM. Placing plates or chambers into an incubator (5% CO2, 37℃) and after culturing more 3 days, then using optical microscope, immunofluorescence cytochemistry (ICC) staining and Western Blot to detect differentiation effects of APDs on CG4 cells.Results: (1) Selection and identification of CG4 cell: CG4 cells originated in newborn rat cerebral cortex in primary culture, and derived from multipotential O2-A progenitor cells. It is an immortalized cell line, which can be unlimited passage. In this study, the CG4 cell line is provided by Manitoba University Neuropsychiatric Institute.①Morphology of CG4 cells under microscope: In the growth medium, the morphology of CG4 cells are similar with oligodendrocytes which have smaller cell type; in the differentiation medium, after 3 days treatment, the morphology were multipolar- shaped structure and similar with the immature and mature oligodendrocytes.②ICC staining results: In the growth medium, PDGFRαprotein was expressed as a marker of oligodendrocyte precursor cells (OPCs) in CG4 cells; in differentiation medium, after 3 days treatment, CNPase and MBP protein were expressed as markers of mature oligodendrocytes in CG4 cells.(2) Toxicity and proliferation effects of APDs on CG4 cells:①Toxicity: Higher concentrations and longer time treatment is toxic for CG4 cells.②Proliferation: there were no proliferation effects of APDs on CG4 cells after 24 h, 48 h and 72 h treatment.③Differentiation: After 3 days APDs inducement, the bipolar CG4 cells decreased, but multipolar shape cells increased. The percentage of processes > 3 group was higher than processes≤3 group both in control and APDs groups.ICC results: After 3 days APDs inducement, PDGFRαexpressed lower and CNPase expressed higher in QUE (0.1μM, 3 d), OLA (1μM, 3 d) and HAL (0.5μM, 3 d) groups.Western blot results:β-actin as a inner parameter, in QUE (0.1μM, 3 d), OLA (1μM, 3 d) and HAL (0.5μM, 3 d) groups, CNPase expressed higher and the difference is remarkable. PDGFRαexpressed lower and the difference of OLA group is prominent. Suggesting these 3 APDs have differentiation effects on CG4 cells.When QUE=0.1μM, OLA=1μM and HAL=0.5μM, compared to control group (QUE/OLA/HAL=0) Olig2 had higher expression. Olig1 of QUE and OLA groups expressed higher and Olig 1 of HLA group also expressed higher but this difference is not marked. The differentiation effects of APDs on CG4 cells may be mediated by Olig1/ Olig2 (bHLH) transcription factors.Conclusions: (1) In GM, CG4 cells morphologically showed a smaller cell bodies and bipolar processes, and expressed PDGFRα, Olig1 and Olig2, which have oligodendrocyte precursor cells characteristic. After 3 days induced in DM, CG4 cells morphologically showed multipolar processes, and expressed CNPase, MBP, Olig1 and Olig2, which have mature oligodendrocytes characteristic. (2) Longer time and higher concentration of QUE (72 h, 100μM), OLA (100μM) and HAL (72 h, 10 and 100μM) treatment is toxic to CG4 cells.(3) The time-effect curve and dose-effect curve showed that there was no proliferation effect of APDs treatment on CG4 cells.(4) QUE (0.1μM, 3 d) and OLA (1μM, 3 d) can promote CG4 cells differentiation, even this effect may be mediated by Olig1/ Olig2 (bHLH) transcription factors.(5) HAL (0.5μM, 3 d) can promote CG4 cells differentiation, even this effect may be mediated by Olig2 transcription factor.The results suggest that, CG4 cells can be used as oligodendrocyte precursor cells in the cell line model. APDs can promote CG4 cells differentiation and the effects may be mediated by bHLH transcription factors.New ideas in this study: To test direct effects of APDs on oligodendrocyte cell line CG4 cells first time in this study. The results in this study provide a certain amount of data to support further explore the mechanism of two types of antipsychotics. The results may in-depth promote exploration the pathogenesis of schizophrenia, and provide theoretical basis and experimental data to better develop efficient new antipsychotic drugs.更多还原