【作者】 杨德雨；

【导师】 谢鹏；

【作者基本信息】 重庆医科大学 ， 神经病学， 2011， 博士

【摘要】 研究背景抑郁症是常见的精神疾病,终生患病率高达16%,其发病机制未明。大量临床和基础研究发现,海马神经发生障碍与抑郁症有关。最新研究发现,抑郁模型小鼠侧脑室下区存在神经发生障碍,提示抑郁症除海马外,成年其他神经发生脑区也可能存在神经发生障碍。嗅球是嗅觉传导的第一中继站,成年哺乳动物嗅球终生进行神经发生以维持嗅觉。嗅球神经发生障碍是否存在于抑郁症发生、发展过程中目前还不清楚,但是许多临床研究发现抑郁症患者嗅觉敏感性降低,并且最新研究发现抑郁症患者嗅球体积减小,提示在嗅球可能存在细胞和分子水平的改变。目的本实验基于前期研究抑郁症与神经发生的基础,应用神经科学的研究思路和方法,观察抑郁大鼠嗅球神经发生情况,进一步探讨神经发生与抑郁症的关系,以及抑郁症患者嗅觉敏感性降低和嗅球体积减小的可能原因。同时观察抑郁大鼠嗅球凋亡情况,多角度探索临床现象的细胞和分子机制。方法本实验分为二部分：第一部分,抑郁大鼠嗅球神经发生的研究；第二部分,抑郁大鼠嗅球凋亡的研究。主要方法和技术路线：1.构建慢性不可预见性温和刺激抑郁大鼠动物模型,并进行嗅觉测试,检测抑郁大鼠嗅觉是否存在障碍。2.运用嗅球连续矢状切片,计算大鼠嗅球体积,检测抑郁大鼠嗅球体积是否减小。3.运用免疫组织化学、western blot等方法,检测抑郁大鼠嗅球未成熟神经元标志物多唾液酸-神经细胞粘附分子(PSA-NCAM)和成熟神经元标志物神经元核抗原(NeuN)阳性细胞数和蛋白表达情况,观察抑郁大鼠神经发生是否存在改变。4.运用western blot、RT-PCR等方法,检测抑郁大鼠嗅球突触前膜功能相关蛋白突触素和突触融合蛋白的表达,观察抑郁大鼠突触前膜功能是否存在障碍。5.运用脱氧核苷酸末端转移酶介导的dUTP缺口末端标记技术(TUNEl)检测抑郁大鼠嗅球凋亡情况,观察抑郁大鼠是否存在凋亡增多。6.运用western blot检测抑郁大鼠嗅球促凋亡蛋白bax和抑凋亡蛋白bcl-2的表达,进一步探讨抑郁大鼠凋亡增多可能的分子机制。结果1.埋藏食物小球法嗅觉测试结果显示抑郁大鼠找到食物时间较正常对照明显延长。2.抑郁大鼠较正常对照嗅球体积减小了21.3%。3.免疫组织化学显示抑郁大鼠嗅球室管膜下区PSA-NCAM阳性细胞数明显减少,western blot进一步印证嗅球PSA-NCAM蛋白表达量减少。4.免疫组织化学显示抑郁大鼠嗅球小球层NeuN阳性细胞数明显减少,western blot进一步印证嗅球NeuN蛋白表达量减少。5.抑郁大鼠嗅球突触前膜功能相关蛋白突触素表达减少,突触融合蛋白表达增多；这两种蛋白mRNA水平并没有发现有明显改变。6.抑郁大鼠嗅球小球层TUNEL阳性细胞数量较正常对照明显增多。7.抑郁大鼠嗅球促凋亡蛋白bax表达增多,抑凋亡蛋白bcl-2表达减少。结论1.抑郁大鼠嗅球神经发生减少,同时伴有突触前膜功能障碍、嗅球体积减小和嗅觉功能降低。嗅球神经发生障碍可能是临床抑郁症患者嗅觉功能减退和嗅球体积减小的原因之一,同时突触前膜功能障碍可能是抑郁患者嗅觉功能减退的分子机制之一。2.抑郁大鼠嗅球凋亡增多。促凋亡蛋白bax增多,抑凋亡蛋白bcl-2减少,可能是其潜在的分子机制之一。更多还原

【Abstract】 BackgroundMajor depression is a common mental disorder and has a lifetime prevalence rates as high as 16%, yet the pathogenesis of this mental disorder is poorly understood. Clinical findings show that hippocampal atrophy exists in the depressed patients, and a variety of evidence has a connection with hippocampal neurogenesis in the pathophysiology of depression. In addition, another adult mammalian brain neurogenesis area, namely, the subventricular zone, was also observed decreased neurogenesis in a mouse model of depression. However, whether other neurogenic regions in the adult central nervous system would be involved with depression is an interesting question. Olfactory bulb (OB) is the first relay station of olfactory transduction pathway, and one of the post-developmental neurogenesis areas in the adult mammalian brain. Reduced OB volume as well as, respectly, olfactory sensitivity was observed in patients with depressive disorder, nevertheless, the molecular and cellular mechanisms is unclear. ObjectiveBased on previous series of studies of depressive disorder, the purposes of present study was to detect neurogenesis and apoptosis in the OB of a rat model of depression by means of neurological idea and methods, and investigate the possible reasons for the reduced OB volume and olfactory sensitivity in depressed patients.MethodsThe experiment consistes of two parts:the first part focuses on the OB neurogenesis in the rat model of depression; the second part concentrates to the apoptosis of the rat model. The main methods and technology:1. Construction of chronic unpredictable mild stress (CUMS) rat model of depression, and then conducting the olfactory test to detecting whether the CUMS rat would exist olfactory dysfunction.2. Calculating the OB volume by means of continuous sagittal slices to detect the alteration of OB volume in the CUMS rat.3. Detecting the number of immature neurons marked by polysialylated neural cell adhesion molecule and mature neurons labeled by Neuronal Nuclei in the OB of rat after 4 weeks chronic stress treatment by means of immunohistochemistry and western blot.4. Detecting the expression of the pre-synaptic proteins synaptophsin and syntaxin in the OB of the rat model by way of western blot and RT-PCR.5. Investigating the apoptosis in the OB of the rat model using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method.6. Detecting the expression of pro-apoptotic protein bax and anti-apoptotic protein bcl-2 in the OB of the rat model furtherly to investigate possible molecular mechanism of the apoptosis.Results1. CUMS rat took significantly longer time to locate the food than control rat.2. Compared with control group, the OB volume of CUMS rat decreased by 21.3%.3. Results of immunohistochemistry and western blot analysis showed a dramatic reduction of immature neurons marked by polysialylated neural cell adhesion molecule in the OB of rat after 4 weeks chronic stress treatment.4. Results of immunohistochemistry and western blot analysis showed a dramatic reduction of mature neurons labeled by Neuronal Nuclei in the OB of rat after 4 weeks chronic stress treatment.5. Data of western blot analysis showed that chronic stress reduced expression of synaptophysin but increased expression of syntaxin in the OB, while significant variation at these mRNA level was not observed. 6. TUNEL-positive cells increased in the glomerular layer in the OB of the CUMS rat.7. Down-regulated expression of bcl-2 protein companying with up-regulated expression of bax protein in the OB of the CUMS rat.Conclusion1. Reduced OB neurogenesis did present in the CUMS rat model of depression. Simultaneously, impaired neurogenesis accompanied with pre-synaptic dysfunction, reduced OB volume and decreased olfactory sensitivity in the CUMS rat model of depression. Reduced OB neurogenesis may be one of the reasons for the reduced OB volume and olfactory sensitivity in the depressed patients, and the pre-synaptic dysfunction may be the underlying molecular mechanism for it.2. Increased apoptosis did exist in the OB of the CUMS rats. Furthermore, up-regulated expression of bax protein and down-regulated expression of bcl-2 protein might function to some extend in the increased apoptosis in the OB of the CUMS rats.更多还原