【作者】 叶仕桥；

【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， 2009， 博士

【摘要】 [背景]在脑组织中14-3-3蛋白十分丰富。14-3-3gamma主要在神经元表达。我们曾报道14-3-3gamma在体外缺血培养的星形胶质细胞中表达上调，并且通过结合磷酸化Bad减少缺血诱导的胶质细胞死亡。[目的]研究14-3-3蛋白在缺血损伤的神经元中的表达，保护作用和机制。[方法和结果]通过Western blot，免疫荧光染色，免疫共沉淀以及原代神经元核转染等方法，我们发现原代培养的小鼠大脑皮层神经元缺血处理后14-3-3gamma表达明显增加。而14-3-3beta，epsilon，eta和zeta在相同处理下没有明显的变化。过度表达14-3-3特异结合蛋白(Diforpein)导致小鼠神经瘤母细胞(N2a)和原代神经元死亡增加。而过度表达14-3-3gamma显著减少缺血诱导的N2a和原代神经元的死亡。除此之外，我们在培养的N2a和原代神经元中加入特异降解14-3-3gamma的siRNA也增加缺血诱导的细胞死亡。为了明确14-3-3gamma在缺血性损伤中的保护机制，通过免疫共沉淀方法，我们发现在原代培养的小鼠大脑皮层神经元经缺血处理后，14-3-3gamma上调并特异结合同时上调的丝氨酸37位点磷酸化beta-catenin，而它与Ask-1没有明显的相互作用。另外，我们还发现在过度表达14-3-3gamma的N2a细胞中，Bax的表达明显下调；而过度表达降解内源性14-3-3gamma-siRNA，Bax的表达增加。在氯化钴诱导的缺氧预适应动物模型中，动物耐受缺氧存活时间明显延长，14-3-3gamma的表达上调，而其它14-3-3亚型发生不同的变化，Bax和p53的表达没有明显影响。[结论]上述结果表明，在缺血神经元14-3-3gamma表达上调并结合磷酸化beta-catenin，降低Bax的表达发挥减轻缺血神经元损伤的重要内源性保护机制，缺氧预适应导致内源性14-3-3gamma上调增加动物耐受缺氧。更多还原

【Abstract】 14-3-3 proteins are abundant in brain tissues and the gamma isoform is mainly expressed in neurons. We have previously reported that 14-3-3gamma could be up-regulated in astrocytes by in vitro ischemia and attenuated ischemia-induced astrocytic death by binding to phosphorylated Bad. In this study, we studied the expression, function and underlying mechanism of 14-3-3gamma in ischemic neurons. The selective up-regulation of 14-3-3gamma in primary cultures of mouse cerebral cortical neurons exposed to oxygen-glucose deprivation (OGD) was demonstrated by Western blot analysis, immunostaining and co-immunoprecipitation methods. Other 14-3-3 isoforms (β,ε,ηandζwere not altered significantly by OGD treatment. Blocking the interaction of 14-3-3 proteins with their ligands by over-expression diforpein (DFP) exacerbated cell death in primary mouse cortical neurons and N2a neuroblastoma cells. Over-expression of 14-3-3gamma enhanced cell survival in OGD-treated neurons and N2a cells significantly while other 14-3-3 isoforms were less effective. Moreover, suppressing 14-3-3gamma expression by siRNA technique facilitated cell death in neurons and N2a cells upon OGD treatment. To study the underlying protective mechanism of 14-3-3gamma, we investigated the interaction of 14-3-3gamma with beta-catenin, Bad, p53 and Ask-1. We found that 14-3-3gamma bound more phospho-s37-beta-catenin in neurons upon OGD incubation while the binding of 14-3-3gamma with Bad, p53 and Ask-1 did not altered evidently. In addition, the expression of Bax was reduced in N2a cells overexpressed 14-3-3gamma while 14-3-3gamma-siRNA increased the Bax level in N2a cells. Finally, in the model of anoxia preconditioning induced by cobalt chloride, we found that the expression of 14-3-3 gamma but not other 14-3-3 isoform was increased in cerebral cortex compared with control. Taken together, these data suggested that 14-3-3gamma-beta-catenin-Bax pathway was an important part of endogenous protective machinery in ischemic neurons. The induction of 14-3-3 gamma may be a protective mechanism of preconditioning.更多还原