【作者】 牛颖；

【导师】 公衍道；

【作者基本信息】 清华大学 ， 生物学， 2009， 博士

【摘要】 阿尔茨海默氏症(Alzheimer’s Disease,AD)是一种神经退行性疾病。它包括两个病理特征:淀粉样斑和神经纤维缠结。淀粉样斑的主要成分是淀粉样肽(Amyloidβ,Aβ),它由淀粉样肽前体蛋白(Amyloid Precursor Protein,APP)在β分泌酶和γ分泌酶切割下产生。本文研究了APP及其水解产物对于细胞容量性钙内流的作用,在此基础上对其作用机制进行了初步探讨。我们的研究结果显示:APP可以降低细胞的CCE。我们用稳定表达野生型人APP695的N2a细胞APPwt与野生型的N2a细胞进行比较,发现APPwt细胞的CCE显著降低,这说明人的APP695会引起细胞CCE的显著降低。我们分别用CCD系统和共聚焦系统证实了该结果。在敲除了APP基因的小鼠胚胎成纤维细胞中的实验证明,鼠的APP同样存在这种作用。我们的实验结果显示,APP分泌到胞外的水解产物sAPP对于细胞的CCE无显著的作用。综合β分泌酶抑制剂和γ分泌酶抑制剂的处理结果,我们推测APP的C端片断可能在CCE的下降中起重要作用。转入C99的细胞的CCE测定结果进一步证实了APP的C端片断会降低细胞的CCE。进一步的研究提示,APP的C端片断与G蛋白的相互作用可能在CCE降低中发挥重要作用。我们首次发现Aβ可以升高细胞的CCE,并发现其作用与Aβ在细胞膜上形成的通道密切相关。在APPswe细胞和APPwt细胞中均证实,Aβ的抗体可以引起细胞CCE的显著降低。这提示,Aβ会升高细胞的CCE。这一作用在γ分泌酶抑制剂的处理结果中得到了证实。用Aβ通道抑制剂氨丁三醇对细胞进行处理,其结果提示,Aβ在膜上形成的通道与Aβ引起的CCE升高密切相关。与此同时,我们在体内和体外水平探讨了Aβ通道抑制剂氨丁三醇在AD治疗中的可能作用。研究结果显示,低浓度的氨丁三醇不会影响细胞的存活。氨丁三醇会通过降低细胞内活性氧的水平减少细胞凋亡,从而起到保护细胞免受Aβ毒性作用的效果。体内的数据显示,氨丁三醇可以升高双转小鼠皮层内磷酸化erk1/2的水平,但其作用和机理还需要进行进一步的探讨。更多还原

【Abstract】 Alzheimer’s disease (AD) is a progressive dementia affecting a large proportion of aging people. AD is characterized by two hallmark brain lesions, i.e. extracellular deposits of amyloidβ(Aβ) and intracellular neurofibrillary tangles. Aβis derived from the processing of Amyloid precursor protein (APP) byβsecretase andγsecretase.In the present study, we focused on the study of the effect of APP and its proteolytic fragments on capacitative calcium entry (CCE). Following are the main content of the thesis:(1) We compared the CCE in N2a cells stably expressing human APP695 (APPwt) with its wild type counterparts, and found that CCE was depressed in APPwt cells. Experiments using CCD and confocal laser scanning microscope got the same results. We can drawed the conclusion that APP depresses CCE. APP knock-out mouse embryo fibroblast cells have an increased CCE compared to its wild type controls, which suggests that mouse APP has the same CCE depression effect as human APP695.(2) Our results showed that sAPPs do not affect CCE.βsecretase inhibitor couldn’t change CCE in APPwt cells, whileγsecretase inhibitor depress CCE in APPwt cells. It’s suggested that C-terminal of APP may involved in APP caused CCE depression. We further demonstrated the role of C-terminal of APP in CCE modulation in N2a cells overexpressing C-terminal fragments of APP. Further experiments suggested that the interaction of APP and G protein may contribute to the APP caued CCE depression.(3) In contrast to APPwt cells, N2a cells stably expressing Swedish mutant APP (APPswe) has a potentiated CCE compared to wild type N2a cells. Anti-Aβantibody can depress CCE in both APPswe and APPwt cells, which suggested that Aβcan potentiate CCE. This conclusion can be also demonstrated inγsecretase inhibitor treatment experiment. It has been reported that Aβforms calcium permeable channels on the cell membrane. We treated the cells with an Aβchannel blocker, tromethamine. The results showed that tromethamine can significantly depress Aβcaused CCE potentiation.(4) We further discuss the potential role of tromethamine in AD therapy. Low concentration of tromethamine can not affect the cell viability. Tromethamine could depress cell ROS, and that may be the way through which it affects cell apoptosis and protects the cells from Aβtoxity. In vivo experiments showed that tromethamine could increase the level of phosphoralate erk protein. The mechanisms need further study.更多还原