成年大鼠去多巴胺能神经元支配的纹状体和胚胎小鼠腹侧中脑发育的蛋白质组学研究

【作者】 沈琰；

【导师】 周嘉伟；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 神经生物学， 2004， 博士

【摘要】 第一部分 帕金森病模型中去神经支配纹状体的蛋白质组学分析本实验对6-羟基多巴胺帕金森病模型中去神经支配纹状体蛋白质组的表达变化进行了研究。我们发现去多巴胺能神经元支配的纹状体提取液具有促进体外培养的胚胎腹侧中脑多巴胺能神经元存活的作用。为了进一步研究这一营养性作用的分子基础,寻找新的促进多巴胺能神经元损伤修复的因子,本实验利用双相凝胶电泳比较分析了去神经支配后纹状体蛋白质组的表达变化。结果显示,纹状体中绝大多数蛋白质的表达水平在去神经支配后没有明显差异,但少数蛋白质的表达水平发生了明显变化:7个蛋白质表达上升,4个蛋白质表达下降。经过基质辅助激光解析电离-飞行时间质谱确定了其中8个蛋白质的种类,表达水平上调的蛋白质包括:淀粉样蛋白前体蛋白2(amyloid precursor-like protein 2, APLP2), 激肽原(kininogen), 葡萄糖激酶(glucokinase), 脑源一型α原肌球蛋白(tropomyosin αchain type brain-1, TMBR-1), 依钙蛋白Ⅰ轻链(calpactin Ⅰ light chain, Calpactin Ⅰ-lc);表达水平下调的蛋白质包括: 神经表皮生长因子样蛋白2(Neural epidermal growth factor-like 2, Nell 2), 甲状腺素受体β-2(thyroid hormone receptorβ-2, THRβ-2),小染色体维持因子6(Mini-chromosome maintenance 6, MCM6)。免疫组织化学染色表明黑质多巴胺能神经元共表达APLP2和酪氨酸羟化酶。6-羟基多巴胺损毁黑质纹状体通路后,APLP2在黑质致密带的表达随着多巴胺能神经元的死亡而降低,但是在去神经支配的纹状体和黑质网状带中APLP2的表达上升。而且,过表达硫酸软骨素修饰APLP2(APLP2-751)的中国仓鼠卵巢细胞系条件培养液具有促进体外培养的胚胎腹侧中脑多巴胺神经元存活的作用,过表达无硫酸软骨素修饰APLP2(APLP2-763)的中国仓鼠卵巢细胞系条件培养液不具有同样的作用。这些结果提示硫酸软骨素修饰APLP2对于黑质多巴胺能神经元可能具<WP=6>有保护和促进损伤修复的作用。更多还原

【Abstract】 During neurodegenerative processes, the injured cells will promote their target tissue to release neurotrophic factors. In this study, we characterized trophic effects of the striatum of adult Sprague-Dawley rat following 6-hydroxydopamine lesions on survival of fetal DA neurons. Treatment of ventral mesencephalic cultures with the striatal extracts delayed DA cell death in dose-dependent manner. In order to better understand the molecular events occurring in the denervated target tissue, we investigated the variations of protein expression in the dopamine-depleted striatum. The rat striatum, ipsilateral to the lesion was analyzed by two-dimensional gel electrophoresis followed by matrix assisted laser desorption/ionization-time of flight mass spectrometry. Seven proteins were up-regulated (188.1-750% compared to control) in response to the lesion, including amyloid precursor-like protein 2 (APLP2), kininogen, glucokinase, tropomyosin α chain, type brain-1 and calpactin Ⅰlight chain; whilst four proteins , including neural epidermal growth factor-like 2, minichromosome maintenance 6, and thyroid hormone receptor β-2, were down-regulated (to between 36% and 59% of levels in sham-operated controls). Three proteins that did not match with available data in the SWISS-PROT protein database were also determined. Immunohistochemical analysis demonstrated colocalization of APLP2 and tyrosine hydroxylase in the nigral neurons. Moreover, <WP=8>reduction of APLP2-positive neurons in the substantia nigra pars compacta as well as the increases in the substantial nigra pars reticulata and in the striatum were observed. Furthermore, the conditioned medium of the Chinese hamster ovary cells over-expressing APLP2-751 (chondroitin sulphate-modified), but not APLP2-763 (nonchondroitin sulphate-modified), was able to increase the number of the tyrosine hydroxylase-positive neurons in fetal mesencephalic cultures. These results suggest that the expression of APLP2, a protein that has been thought to be associated with Alzheimer’s disease, is up-regulated in the striatum following dopaminergic denervation. They also support the view that chondroitin sulphate-modified APLP2 protein may play an important role in protection and repairation of dopaminergic neuron.更多还原