【作者】 李慧；

【导师】 刘剑峰；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 神经细胞死亡在各种神经退行性疾病中是重要的病理特征，大量神经细胞的死亡会导致神经系统出现病变。在神经系统病变状态下研究其适应性和抵抗性具有非常重要的实际意义。神经系统疾病，如神经退行性疾病或脑缺血和内质网应激反应，具有一定的相关性，成纤维细胞生长因子2或碱性成纤维细胞生长因子（Fibroblastgrowth factor2/Basic fibroblast growth factor, FGF2/bFGF）是一种多效生长因子，在神经系统中能够促进神经前体细胞增殖和生长及诱导细胞分化。研究发现，在脑受损后FGF2表达明显增加，通过激活PI3K/AKT, ERK等信号通路促进神经细胞的存活。FGF2是否影响内质网应激诱导的凋亡效应目前尚无相关报道。蛋白Nck是一种无催化活性的酪氨酸激酶的接头蛋白，广泛介导活化的受体酪氨酸激酶与下游靶蛋白之间的信号传递，但Nck在FGF2诱导的保护效应中的作用机制还有待进一步阐明。本文以小鼠神经干细胞C17.2为研究对象，利用衣霉素(Tunicamycin, TM/Tun)作为一种内质网应激反应化学诱导剂模拟病理条件，诱导细胞发生凋亡，进而针对FGF2对衣霉素诱导细胞凋亡的保护效应及Nck在该效应中扮演的角色进行研究。我通过检测通路中相关蛋白发现，FGF2显著抑制了Tun诱导细胞凋亡蛋白PARP的剪切，但高表达Nck会拮抗FGF2的这种效应。FGF2促进AKT和ERK的活化，但高表达Nck对FGF2诱导的AKT和ERK存在不同的影响，表现在显著抑制FGF2促AKT的活化，但对ERK活化不存在显著影响。当下调Nck时，FGF2促AKT活化的程度增加。高表达不同SH3结构域突变的Nck不改变FGF2促AKT活化的规律，表明Nck影响FGF2促AKT活化的效应依赖于SH3结构域的完整性。此外免疫沉淀结果表明，Nck与RasGAP之间存在组成性的相互作用，推测Nck借助与RasGAP的互作调节Ras和AKT的活性。这些结果表明，FGF2对内质网应激下的神经细胞具有明显的保护效应，接头蛋白Nck作为FGF2信号通路的下游接头蛋白分子，招募形成的蛋白复合体会在FGF2作用下，通过影响RasGAP来调控Ras的活性进而调节PI3K/AKT通路，最终决定神经细胞的生存能力。本文的研究结果为进一步深入揭示FGF2促神经细胞的生存机制奠定了基础，同时有望为相关神经退行性疾病的治疗提供新的理论基础和药物研发的新靶点。更多还原

【Abstract】 The survival of neuron is frequently used as the pathological feature in variousneurodegenerative diseases and death of a great many neurons can seriously influencenervous system function. Therefore, it’s of great significance to explore the resistance andadaptability mechanisms in nervous system under pathological state. Recent researcheshave found that endoplasmic reticulum stress signal pathway has close relationship withcentral nervous system diseases, including neurodegenerative diseases and regionalcerebral ischemia. Fibroblast growth factor2or basic fibroblast growth factor is amultiple-effect factor, having close relationship with neurodegenerative diseases, canpromote cell proliferation and growth, induce cell differentiation and so forth. Evidenceshave confirmed that increasing in FGF2expression after brain injury can promote neuronsurvival through PI3K/AKT and ERK activation. There is no related report whether FGF2affects ER stress induced apoptosis. Adaptor protein Nck is a kind of non-catalytic proteinkinase and widely conducts activated receptor tyrosine kinases and downstream proteinssignal transduction, however there is little known about mechanism of Nck in FGF2induced protective effect, which needs further clarified.This study used murine nervous stem cell C17.2as research object and tunicamycin,which is a chemical induction of ER stress, to imitate pathological condition and figureout the protective effect of fibroblast growth factor2and roles Nck plays in. Throughdetecting related proteins in signaling pathway, we observed that FGF2notably inhibitedapoptotic protein PARP cleavage induced by Tun.However overexpression of downstreamprotein Nck could antagonize FGF2-mediated effect. FGF2could activate AKT and ERKactivation, but Nck overexpression existed different influences on AKT and ERKactivation. It could remarkably inhibited FGF2-promoted AKT activation but not ERK.When knockdown Nck, there was a increasing in FGF2-induced AKT activation.Overexpression different SH3domain mutants in Nck wouldn’t change FGF2-inducedAKT activation indicating Nck affects FGF2-induced AKT activation is dependent on the SH3domains integrity. What’s more,immunoprecipitation result showed that Nck and RasGAP have constitutive interaction,suggesting Nck regulates Ras and AKT activities with the help of interaction withRasGAP.The results showed that FGF2can protect C17.2from tunicamycin induced ER stress,and Nck as a downstream adaptor protein in FGF2signaling pathway is able to recruitvarious proteins to regulate PI3K/AKT pathway. Moreover, Nck can interact with RasGAPto regulate Ras activity. This research may lay a foundation for further revealingmechanism of FGF2-induced neuron pro-survival, and probably provide new targets fordrug design and development for neurodegenerative diseases.更多还原