【作者】 李莉；

【导师】 陈晓钎；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2013， 博士

【摘要】 在中枢神经系统中，神经元突起生长对神经回路和突触连接的建立是必不可少的。诱导神经元突起生长对于中风、神经退行性疾病、外伤性中枢神经系统损伤等疾病的治疗十分重要但却十分困难。神经元成熟后失去产生突起的能力，衰老神经元突起再生尤其困难。因此，为了寻找有效的药物或改进治疗手段来治疗神经系统疾病，必需清楚地了解调节突起生长的分子机制。脑红蛋白（neuroglobin，Ngb）作为一个具有高氧亲和力的组织珠蛋白，在脊椎动物中枢和周围神经系统的神经元中广泛并特异性表达。在过去大量的研究表明Ngb高表达对缺氧/缺血诱导的神经元损伤及脑缺血动物模型具有保护作用。迄今为止，国内外还未见有关Ngb与神经元突起生长相关性的报道。本研究采用小鼠成神经瘤N2a细胞系和原代皮层神经元作为实验材料和研究对象，利用细胞培养、分子生物学、免疫印迹和细胞形态学等研究技术和方法，进一步探讨了Ngb对神经元突起生长的作用及其潜在的分子机制。在小鼠成神经瘤N2a细胞中，采用RNA干扰技术抑制Ngb内源性表达后细胞呈现出显著的突起缺陷表型。反之，过表达Ngb后Ngb基因编码的蛋白表达量增加，神经元的突起明显增长。我们进一步在原代培养的大鼠大脑皮层神经元中发现，随神经元发育，突起的增长，Ngb基因表达量显著增加，而脑红蛋白的同源基因细胞红蛋白（cytoglobin,Cygb）的基因表达没有变化。在原代培养的大鼠大脑皮层神经元中过表达Ngb神经元的突起明显增长。反之，抑制原代培养的大鼠大脑皮层神经元中内源性Ngb的表达，神经细胞的突起与对照组相比明显变短。药理学研究发现在原代皮层神经元中Ngb促进突起生长的作用依赖于Akt及p38MAPK蛋白的活性。我们进一步在撤血清不同时间点观察Ngb对PI3K/Akt及p38MAPK信号通路的影响，结果显示在撤血清处理9hr的小鼠成神经瘤N2a细胞中过表达Ngb明显上调了Akt及p38的磷酸化水平，同时下调了PTEN的磷酸化水平。双分子荧光互补实验（BiFC）和GSTpull-down结果进一步证实Ngb及其突变体（E53Q,E118Q,K119N,H64A,H64L,Y44D)与EGFR、Akt、p38MAPK和PTEN发生差异性的结合。NgbE53Q这一突变体与野生型Ngb相比同EGFR、Akt和p38MAPK的结合增加，而同PTEN的结合减少，使其对PTEN及Akt的磷酸化水平的调控更为明显，从而增强了Ngb对突起生长的诱导作用。最后我们在小鼠成神经瘤N2a细胞中过表达Ngb后再予无氧无糖(体外模拟缺血)处理，细胞的突起与对照组相比明显增长，证明增加Ngb在缺血病理状态下仍具有促进神经元突起生长的作用。在脑卒中、老年痴呆、脑/脊髓损伤等疾病中，神经元突起的断损、缩短是脑功能障碍的重要原因，而促进神经元突起的生长或再生是治疗以上脑疾病的关键。因此，Ngb促进神经元突起生长这一新功能为开发治疗神经系统疾病的药物提供了基础。更多还原

【Abstract】 During the development of the central nervous system (CNS), neurite outgrowth is required for neuronal path findings and the establishment of synaptic connections. Inducing neuritogenesis is a crucial but tough step in treating diseases such as stroke neurodegenerative diseases or traumatic CNS injury. Along with aging, regenerating neurite is of particular difficult because neurons lost their ability to produce neurite after maturation. Understanding clearly the molecular machinery regulating neurite outgrowth is essential for searching effective drug or improving treatments for various neurological diseases.Neuroglobin (Ngb), a neuronal-or brain-specific respiratory protein, is the third heme-containing globin discovered in mammalians. Most studies have shown that overexpression of neuroglobin exerts neuroprotection against neuronal hypoxia/ischemia-induced damage in cultured neurons and in cerebral ischemic animal models. However, little is known about its role in neuritogenesis.To investigate the roles and the potential mechanisms of neuroglobin on neurite outgrowth, mouse neuroblstoma N2a cells and primary cultures of cerebral cortical neurons were applied. Respectively, cell culture, molecular biology technology, immunoblotting and cytomorphological methods were performed for this study.Knocking-down of Ngb exhibited a prominent neurite-deficient phenotype in neuroblstoma N2a cells. Silencing Ngb prevented neurite outgrowth while overexpression of Ngb but not homologous cytoglobin (Cygb) promoted neurite outgrowth upon serum deprivation. In primary cultures of cerebral cortical neurons, Ngb was up-regulated along with culture ages. Overexpression of Ngb but not Cygb promoted neurite outgrowth while knocking-down of Ngb reduced neurite outgrowth in primary cultures of cerebral cortical neurons. Pharmacological studied revealed that Ngb-promoted neurite outgrowth was depended on activities of p38MAPK, Akt but not protein kinase A (PKA), Ca2+/Calmodulin-Dependent Protein Kinase Ⅱ (CaMKII) or the Erk in cortical neurons. Western blots results show that overexpression of Ngb suppressed expression of p-PTEN, enhanced expression of p-AKT and p-p38in N2a cells after withdrawl serum9hr. Bimolecular fluorescence complementation and glutathione S-transferase pull-down assays revealed that neuroglobin and various mutants (E53Q, E118Q, K119N, H64A, H64L, Y44D) bound with EGFR, Akt, p38MAPK and PTEN differentially. Neuroglobin E53Q mutant showed a prominent reduced PTEN-binding but increased Akt-binding, resulting in decreased p-PTEN, increased p-Akt and increased neurite length. Further, overexpression of Ngb promoted neurite outgrowth in N2a during hypoxia. Taken together, our data suggest a physiological and pathological function of Ngb in neuritogenesis in the brain, providing a potential therapeutic target for the axonopathy in neurological diseases.更多还原