RNA干扰结合慢病毒载体过表达研究Gnaq基因在神经细胞氧化应激中的作用及分子机制

The Gnaq protein (Guanine nucleotide binding protein alpha q polypeptide), which is coded by the corresponding Gnaq gene, is a member of the α-subunit multigene family of G-proteins. Gnaq is abundant in brain tissue. In our previous study, we found the expression level of Gnaq was reduced with aging in the forebrain of the SAMP8 mice. It is still unknown whether and how Gnaq play protecting roles in the degeneration diseases in central nervous system (CNS). Damage of oxidative stress is a main factor resulting in the occuring and development of CNS degeneration diseases. Documents indicate that there are complicated internal links among Gnaq, oxidative stress, PKC signal pathway and abilities of learning and memory. However, it is very little that we know about the underlying mechanisms in these links. "Whether Gnaq protects nerves from degeneration by promoting the anti-oxidative-damage capacity of neural cells？" "Which signal pathways participate in the anti-oxidative-damage function of Gnaq?" and " What kind of crosstalking exist among the probable related signal pathways?". All of these questions are waiting for being answered. By combining RNA interference with lentivector overexpression techniques, the present study aims to explore whether Gnaq can enhance the anti-oxidative-damage property of neural cells, and to analyze the molecular mechanisms how Gnaq improve the anti-oxidative-damage property of neural cells, to probe the signal pathways which are probably modulated by Gnaq. Furthre more, proteomics profiling and bioinformatics approaches will be performed to analyze the Gnaq-overexpressed neural cells so that the potential crosstalking among related signal pathways would be found. In the present study, we will copy the Gnaq gene by PCR, and then clone it into the lentivirus vector PLIG to get a recombinant plasmid. Then we will amplify plasmids, produce self-inactivating lentivirus and transfect them into target cells. The ability of Gnaq-overexpressed neural cells resisting oxidative stress will be determined by MTT experiments. The concentration of active oxigen and activity of mercaptan antioxidant enzyme will also be measured. Combining with all of these techniques, we will try to illuminate the probable molecular mechanisms of Gnaq in oxidative stess of neural cells, and to provide a basis for designing new strategy in prevention and treatment of CNS degeneration diseases.

Gnaq是G蛋白α亚单位多基因家族的一员，由相应的Gnaq基因所编码,富含于脑组织中。我们前期研究发现SAMP8小鼠额叶内Gnaq的表达水平随衰老明显下降。氧化应激损伤是中枢神经退行性疾病发生发展的重要促进因素。文献表明，Gnaq、氧化应激、PKC通路、学习记忆能力之间存在复杂的内在联系，但对其确切机制知之甚少。"Gnaq是否通过增强神经细胞抗氧化损伤能力而发挥抗神经退行性变的作用？"、"Gnaq通过哪些信号通路而发挥抗氧化应激损伤的作用？"、"相关信号通路之间存在怎样的分子交联？"等问题亟待解决。本研究旨在利用小RNA干扰技术结合慢病毒载体过表达技术，研究Gnaq是否能增强神经细胞抗氧化应激损伤的能力，分析Gnaq影响神经细胞抗氧化应激损伤能力的分子机制，探讨可能受Gnaq调控的信号转导通路，并通过蛋白组学技术结合生物信息学技术，研究可能受Gnaq调控的信号转导通路之间的分子交联。

Gnaq是G蛋白α亚单位多基因家族的一员，富含于脑组织中，研究发现小鼠前皮质内Gnaq表达水平随衰老明显下降，而几乎所有与衰老相关的神经退行性疾病，都与氧化应激有着密切的联系。本课题的主要研究内容包括：（1）实现Gnaq基因在神经细胞中沉默或过表达；（2）研究Gnaq基因在神经细胞抗氧化损伤中的作用；（3）研究Gnaq基因发挥抗氧化损伤作用可能涉及到的信号通路；（4）研究Gnaq基因的靶向分子和所涉及信号通路之间可能存在的交联。课题研究主要获得了以下结果：（1）成功构建了持续、稳定过表达Gnaq的细胞株Gnaq-SY5Y和对照细胞Vector-SY5Y；（2）发现Gnaq-SY5Y细胞的增殖速率明显快于Vector-SY5Y细胞；（3） Gnaq-SY5Y细胞的抗氧化损伤能力比Vector-SY5Y细胞明显增强，荧光探针及流式细胞检测显示Gnaq-SY5Y细胞内的基础活性氧含量低于Vector-SY5Y细胞，H2O2氧化刺激后，Gnaq-SY5Y细胞活性氧含量增幅也低于Vector-SY5Y细胞，而Gnaq-SY5Y细胞内的NF-κB/p65和磷酸化的Erk1/2明显低于Vector-SY5Y细胞，在H2O2氧化刺激后，Gnaq蛋白的表达水平显著下降，NF-κB/p65和磷酸化的Erk1/2的表达水平则显著升高；（4）通过双向凝胶电泳技术筛选了Gnaq-SY5Y细胞内的50个明显差异表达蛋白，其中37个蛋白质上调，13个蛋白质下调。结合质谱分析发现其中的38个蛋白质在数据库中有相应的记录，涉及氧化、炎症、凋亡、自噬、线粒体等多方面的功能及相关信号通路。上述结果说明：(1) Gnaq蛋白能增强神经细胞活力，能减少氧化应激对神经细胞造成的损伤；（2）Gnaq蛋白可能通过影响NF-κB信号通路和MAPK/Erk信号通路，减少神经细胞内活性氧的产生，增强神经细胞对活性氧的清除能力，从而发挥抗氧化应激损伤的作用；（3）过表达Gnaq后，引起了细胞内多种蛋白质表达量的改变，涉及氧化、炎症、凋亡、自噬、线粒体等多方面的功能及NF-κB、MAPK等多条信号通路调控的变化，提示Gnaq可以通过影响多条信号通路而发挥增强神经细胞活力和抗氧化损伤能力的作用，Gnaq可能是这些通路交联的关键点之一。