Pur alpha蛋白对淀粉样前体蛋白基因表达调控机制的研究

The Alzheimer's disease was first described in 1906 and was identified as an ''unusual disease of the cerebral cortex'', which caused memory loss, disorientation, hallucinations. The most important hallmark in Alzheimer's disease study was the establishment of amyloid hypothesis (Amyloid beta, A beta). The A beta aggregates on the CNS to form the senile plaque which disrupts the brain cells, clogs the points of cell-to-cell communications,activates immune cells that trigger the inflammation and devours the disable cells and ultimately, kills cells. In our previous study, we found that the Pur a protein can down-regulate the amyloid precursor protein (APP) promoter activity. In order to better understand the mechanism of this negative regulation of Pur alpha, we use the luciferase contructs which contains the APP promoter, to checked the transcriptional regulation of Pur alpha on APP gene expression through the analysis of transcriptional activation. We focus the target on the 5'UTR where the predicted APP binding site and Egr-1 binding site are overlapped. We promote a hypothesis that the regulational effects of Pur alpha on APP promoter is completed through another transcriptiona factor, Egr-1, which was proved to be a positive rehulational factor on APP promoter in our previous studies.Since the binding sites of these two proteins on APP promoter is overlapped,there might be a displacement mechnism for the regulation of APP gene expression. In order to prove our hypothesis, we put the stress on the interaction of the two proteins. A serious approches will be employed to study the physical interactions of these two proteins in vitro and in vivo, also the functional interaction will also be evaluated.The experiment will be set as following steps, first to confirm the physical binding site of these two proteins on APP promoter 5'UTR area and the thranscriptional activity will be assessed by the luciferase activity analysis,appropriate mutations will be created to confirm the function of the binding and also, the EMSA, CHIP's assay and DNA foot proting approches will be used for the further confimation.Second the interaction of these two proteins will be evaluated through a series approches including the pull down assay and co-IP/wester, colocalization as well as the subcellular distributions of the two proteins, the functional interaction domain will be assessed through the series deletions of the Pur alpha and Egr-1; Last step will be engaged the changes of Amyloid peptide within the cells and the effects of Pur alpha on secretase and apopain,cell cycle and cyclins will be also evaluated. Above all, we will explain the mechanism by which the Pur alpha regulate the APP gene expression privide the clues for the treatment of AD.

阿尔茨海默病（AD）是一种渐进性中枢神经变性型疾病，并伴有由淀粉样蛋白β多肽在细胞外形成老年斑等神经病理学方面的损害。Aβ是淀粉样前体蛋白（APP）异常裂解的产物，其表达水平对淀粉样蛋白多肽的形成和在神经组织中的沉积有着密切的关系。本项目拟研究APP基因表达的转录调控，将采取含有APP启动子的萤火虫荧光素酶的报告质粒在神经母细胞瘤U87MG细胞中的转录活性分析的方法来探讨人转录活化因子Purα对APP基因负调控的机制，并通过Pur α和Egr-1蛋白的相互作用的研究分析，以期阐明Purα对APP蛋白表达的调控作用的机制，并进一步探讨蛋白与蛋白，蛋白与核酸之间的相互作用， 揭示转录因子Pur α在调控蛋白表达过程中的作用机制，从而为AD的防治提供一条新途径。同时通过对DNA-蛋白，蛋白-蛋白相互作用的研究，进一步探索基因表达的调控方面的机制，为AD的治疗提供新的思路。

阿尔茨海默病（AD）是一种渐进性中枢神经变性型疾病，并伴有由淀粉样蛋白β多肽在细胞外沉积形成老年斑（senile plaque）和陶蛋白在细胞内形成的神经纤维缠结(NFTs)等特异性神经病理学方面的损害。Aβ是淀粉样前体蛋白异常裂解的产物，其表达水平对淀粉样蛋白多肽的形成和在神经组织中的沉积有着密切的关系。人转录活化因子α (Purα) 是一种转录调控因子，它在DNA复制，转录，细胞周期调控，DNA损伤的修复等许多生命环节中发挥着重要的作用。.本研究采取将含有APP基因启动子的报告质粒与Purα真核表达载体共转染到U87MG, U251以及HT22中的转录活性分析的方法来检测Purα蛋白对APP基因表达的负调控机制；同时在APP基因启动子上确定与Purα蛋白相结合的位点，然后通过EMSA和ChIP’s Assay来确定该位点的正解性，并通过基因突变的方法切除该位点，检测该位点切除后Purα蛋白对APP基因表达的调控作用；在我们的研究分析中还发现早期生长反应蛋白-1对APP基因表达有正调控作用，通过对Purα和Egr-1蛋白相互作用的研究分析，进一步阐明Purα蛋白对APP基因表达的负调控的机制，通过蛋白与蛋白，蛋白与核酸之间的相互作用，揭示转录因子Purα在调控蛋白表达过程中的作用机制；此外我们还构建了不同的含有Purα核酸结合区域的缺失突变体，更进一步确定了Purα蛋白不同的功能结构域在调节APP基因表达中的功能；与此同时，我们还探讨了Purα蛋白对BACE1基因表达的调控作用，以进一步确定Purα蛋白在AD防治中的作用。我们的实验结果表明，Purα蛋白对BACE1的基因表达同样有负调控作用，二者结合起来更加证实了Purα蛋白对AD的可能的预防和治疗作用。.进一步的扩展研究我们还观察了Purα蛋白对退行性神经病变的研究，主要集中在Purα蛋白在DNA损伤的修复方面的研究。我们利用HU处理小鼠海马神经元细胞造成DNA损伤的模型，然后对Purα蛋白对其保护作用进行了较为深入的研究。另外还在动物模型上探讨了Pura蛋白对癫痫所造成的DNA损伤的保护作用，这些结果表明，Pura蛋白在维持神经系统基因组DNA的稳定性方面有着明显的作用。