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標題: | 探討白芨多醣體降低類澱粉蛋白之細胞毒性及其在預防及治療阿茲海默症之效果 The Effect of Bletilla Striata Polysaccharide on Reducing Aβ-induced Cytotoxicity for Alzheimer's Disease Prevention and Therapy |
作者: | Hong-Hsiang Liao 廖泓翔 |
指導教授: | 林?輝 |
關鍵字: | 阿茲海默症,類澱粉蛋白,白芨,白芨多醣體,β-secretase, Alzheimer’s disease,Amyloid-β,Bletilla Striata,Bletilla Striata polysaccharide,β-secretase, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 阿茲海默症為一種常見的神經退化疾病,是造成失智症的主要原因之一。類澱粉蛋白(Amyloid-β) 被視為造成阿茲海默症的起因之一,其由前類澱粉蛋白 (Amyloid precursor protein) 被 β-secretase 與 γ-secretase切割後所產生。當類澱粉蛋白聚集於大腦時,會造成氧化壓力 (oxidative stress)、慢性發炎反應 (chronic inflammatory response) 以及神經突觸功能喪失 (synaptic dysfunction),導致大腦神經退化與死亡,而目前臨床上尚未有能夠治療阿茲海默症的藥物。白芨多醣體已被證實具有的抗氧化、抗發炎與促進細胞增殖的特性,本研究希望能夠藉由白芨多醣體之特性降低類澱粉蛋白所造成的細胞毒性,並進一步探討白芨多醣體對 β-secretase 表現量之影響,以達到預防及治療阿茲海默症。
本研究以 FTIR 與 NMR 確認萃取出白芨多醣體的官能基與結構;透過ThT assay、Western Blot 與 TEM 觀察類澱粉蛋白纖維與寡聚醣的形成。體外實驗的部分,依據 ISO 10993 規範,由WST-1 測試確認其生物相容性;DCFDA 與 RT-PCR 確認抗氧化與抗發炎效果;Live and Dead 觀察白芨多醣體抑制類澱粉蛋白產生細胞毒性之能力。體內實驗部分,行為實驗水迷宮評估老鼠的記憶能力;Western Blot評估白芨多醣體抑制 β-secretase表現量之能力;組織切片染色呈現海馬迴與大腦皮質的細胞與組織之狀況;免疫組織化學染色觀察海馬迴與大腦皮質中類澱粉蛋白的多寡與分布。 根據目前的實驗結果,體外實驗中,我們已成功證實白芨多醣體能夠預防與治療類澱粉蛋白所誘發的氧化壓力與發炎反應,有效降低類澱粉蛋白所造成的細胞毒性。體內實驗中,我們已成功證實白芨多醣體能夠有效減緩氯化鋁誘導之阿茲海默症老鼠的症狀,藉由海馬迴與大腦皮質中的損壞,降低記憶能力的喪失,也藉由降低 β-secretase 的表現量,以減少類澱粉蛋白的產生與斑塊之堆疊。 Alzheimer’s disease (AD) is the primary cause of age-related dementia. Amyloid-β (Aβ) is regarded as one of the reasons of AD. Amyloid precursor protein is cleaved by β-secretase and γ-secretase to produce amyloid-β, whose aggregation implicates in neuronal degeneration and cognitive decline in AD. It induces oxidative stress, chronic neuroinflammation, and synaptic dysfunction, leading to the death of neurons in the brain. Nowadays, there is no effective therapy to cure AD in clinical treatment. Bletilla Striata polysaccharide (BSP) has been proved to have anti-oxidative, anti-inflammatory effects and be able to induce cell proliferation. Therefore, the purpose of this study is to utilize the characteristics of BSP to reduce Aβ-induced cytotoxicity. Further, we explore the effect of BSP on β-secretase levels to achieve the prevention and treatment of AD. In this study, FTIR and NMR are used to confirm the functional groups and chemical structure of BSP. ThT assay, Western blot and TEM are used to observe the formation of Aβ fibrils and oligomers. WST-1 assay is used to confirm the biocompatibility of BSP. DCFDA and RT-PCR tests are used to confirm the anti-oxidative and anti-inflammatory effects of BSP. Live and Dead test is used to verify the ability of BSP to reduce the cytotoxicity of Aβ. Morris Water Maze is performed to evaluate the retention of working and spatial memory in rats. Western Blot is used to evaluate the ability of BSP to decrease β-secretase levels. Section of cortex and hippocampus tissues are stained with hematoxylin and eosin to observe the cells and the tissues. Immunohistochemical staining of the section of cortex and hippocampus tissues is used to show the distribution of amyloid-β. The blood test is used to evaluate the biochemistry of blood. The current results had successfully proven that BSP could reduce the cytotoxicity of amyloid-β through the treatment and prevention of the oxidative stress and inflammation induced by amyloid-β. Furthermore, reducing in the damage of hippocampus and cortex lead to attenuate the neurobehavioral impairments by treating BSP in AlCl3-induced Alzheimeric rats. Meanwhile, the evidence of greatly reducing in β-secretase levels and the deposit of amyloid-β plaque were found in the BSP treated rats. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72881 |
DOI: | 10.6342/NTU201901622 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學工程學研究所 |
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