β-淀粉样蛋白单体和寡聚体在星形胶质细胞中的内吞、转运及降解机制的探索与比较

发布时间：2019-07-05 06:54

【摘要】：阿尔茨海默症(AD)是一种常见的具有高发病率、高患病率及高致残率的神经退行性疾病。许多研究证明β-淀粉样蛋白寡聚体（oAβ）具有极大的细胞毒性，能够促使神经元死亡，导致AD发生。星形胶质细胞倾向于清除oAβ，但是机制仍不清楚。因此，本文以星形胶质细胞瘤U87细胞系为实验对象，通过体外制备oAβ_(42)并采用硫磺素T（ThT）染色和透射电镜对其表征，利用激光共聚焦荧光显微技术，研究β-淀粉样蛋白单体（sAβ）和寡聚体在星形胶质细胞上的内吞、转运及降解机制的差异。胞吞实验结果表明，，sAβ_(42)和oAβ_(42)均能通过巨胞饮途径进入星形胶质细胞，而且胆固醇在它们的内吞中发挥重要作用，但是统计数据表明二者对同一种胞吞途径的依赖程度不同。同时，经典的网格蛋白介导的胞吞途径并不参与这两种形态多肽的内吞过程，但是dynamin2的剪切作用却是此过程必不可少的。此外，利用RNA干扰技术沉默脂蛋白受体相关蛋白1（lipoprotein receptor-related protein1, LRP1）则抑制了sAβ_(42)的内吞过程，但oAβ_(42)的内吞不受影响。以上结论表明星形胶质细胞内吞sAβ_(42)和oAβ_(42)的机制存在差异，而且数据分析显示其更倾向于吸收oAβ_(42)。共定位实验结果表明sAβ_(42)和oAβ_(42)在胞内转运的早期会进入早期内涵体，但最终则在溶酶体中积累。通过细胞活力测定实验（MTS）和体外降解实验进一步证实星形胶质细胞具备降解sAβ_(42)和oAβ_(42)的能力，及时避免了oAβ_(42)的细胞毒性，然而，与sAβ_(42)相比，oAβ_(42)却更难以被星形胶质细胞降解。因此，我们的研究是首次探究并比较了sAβ_(42)和oAβ_(42)在星形胶质细胞上的内吞、转运及降解机制的差异，为AD的预防和治疗提供新的靶点。
文内图片：

图片说明：Aβ42寡聚体的形成和表征(A)oAβ42透射电镜显微照片取oAβ42样品吸附在聚乙烯醇缩甲醛包被的铜网上，室温静置2分钟后，去掉多余的液体
[Abstract]:Alzheimer's disease (AD) is a common neurodegenerative disease with high incidence, high prevalence and high disability rate. Many studies have shown that 尾-starch protein oligomer (oA 尾) has great cytotoxicity, which can promote neuronal death and lead to AD. Astrocytes tend to eliminate oA 尾, but the mechanism is still unclear. Therefore, oA 尾 _ (42) was prepared in vitro and characterized by sulfur T (ThT) staining and transmission electron microscope. The difference of endocytosis, transport and degradation mechanism between sA 尾 and oligomer on astrocytes was studied by laser confocal fluorescence microscopy. The results of endocytosis test showed that both sA 尾 _ (42) and oA 尾 _ (42) could enter astrocytes through the megalocytic drinking pathway, and cholesterol played an important role in their endocytosis, but the statistical data showed that both of them depended differently on the same endocytosis pathway. At the same time, the classical grid protein mediated endocytosis pathway is not involved in the endocytosis process of these two forms of peptides, but the shear effect of dynamin2 is essential to this process. In addition, silencing lipoprotein receptor associated protein 1 (lipoprotein receptor-related protein1, LRP1) by RNA interference technique inhibited the endocytosis of sA 尾 _ (42), but the endocytosis of oA 尾 _ (42) was not affected. These results suggest that there is a difference in the mechanism of endocytosis of sA 尾 _ (42) and oA 尾 _ (42) in astrocytes, and the data analysis shows that oA 尾 _ (42) is more likely to be absorbed by astrocytes. The results of co-localization experiments showed that sA 尾 _ (42) and oA 尾 _ (42) entered the early stage of intracellular transport, but eventually accumulated in lysosome. (MTS) and in vitro degradation experiments further confirmed that astrocytes had the ability to degrade sA 尾 _ (42) and oA 尾 _ (42), and the cytotoxicity of oA 尾 _ (42) was avoided in time. However, compared with sA 尾 _ (42), oA 尾 _ (42) was more difficult to be degraded by astrocytes. Therefore, our study is the first time to explore and compare the difference of endocytosis, transport and degradation mechanism between sA 尾 _ (42) and oA 尾 _ (42) in astrocytes, so as to provide a new target for the prevention and treatment of AD.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

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