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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 林君榮(Chun-Jung Lin) | |
dc.contributor.author | Chih-Yu Lee | en |
dc.contributor.author | 李之瑀 | zh_TW |
dc.date.accessioned | 2021-07-11T15:21:23Z | - |
dc.date.available | 2024-03-11 | |
dc.date.copyright | 2019-03-11 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-02-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78815 | - |
dc.description.abstract | 神經發炎是許多腦部相關疾病的共同病徵,而自體抗發炎物質腺苷其胞外濃度的變化已知對於調控發炎的嚴重程度具有一定影響力。其中,第一型及第二型平衡型核苷轉運蛋白 (equilibrative nucleoside transporters, ENTs) 是腦內主要負責調控細胞內外腺苷運輸及恆定的膜蛋白。由於我們的研究發現ENT2於腦部的表現量明顯高於ENT1,因此透過CRISPR-Cas9技術產製出ENT2基因剔除小鼠作為本實驗主要使用的動物模型,嘗試探討ENT2對於由脂多糖所引起的神經發炎所扮演的角色。研究結果發現ENT2基因剔除可以使得因脂多糖所引起細胞激素 (cytokine) 之mRNA表現量增加、星狀膠細胞增生 (astrogliosis)、微膠細胞活化程度提高等發炎反應都獲得顯著緩解。此外,腦部微透析的結果亦顯示相較於littermate小鼠而言,ENT2基因剔除鼠具有較高的胞外腺苷濃度,並會因為多劑脂多糖的刺激產生上升趨勢。綜上所述,我們的研究指出ENT2對於維持腦部胞外腺苷恆定及影響腺苷調控之神經保護機制中佔有很重要的角色。 | zh_TW |
dc.description.abstract | Neuroinflammation is a common characteristic of many brain related disorders. Adenosine is an endogenous anti-inflammatory agent and its extracellular level in the brain may play an important role in modulating the status of neuroinflammation. The equilibrative nucleoside transporters, ENT1 an ENT2, are the major membrane transporters responsible for cellular uptake of adenosine and the maintenance of extracellular adenosine level in the brain. Given that the expression of ENT2 is higher than that of ENT1 in the brain of mice, ENT2 knockout (KO) mice were established by CRISPR-Cas9 technique and used to clarify the roles of ENT2 in modulating neuroinflammation induced by the treatment of lipopolysaccharide (LPS). The results showed that ENT2 KO significantly ameliorated the LPS-induced neuroinflammation, in terms of the increase of cytokine levels, astrogliosis and microglia activation. The results of in-vivo brain microdialysis study confirmed that the basal level of extracellular adenosine were higher in ENT2 KO mice than that in the littermate controls. Moreover, multiple LPS treatments caused a significant increase in brain adenosine level in ENT2 KO mice, but not in the littermate controls. Taken together, these findings suggest that ENT2 play a crucial role in maintaining extracellular adenosine and adenosine-mediated neuroprotection in LPS-induced neuroinflammation. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T15:21:23Z (GMT). No. of bitstreams: 1 ntu-108-R05423020-1.pdf: 2403770 bytes, checksum: f72810a7c630091f1cac5a0b25b5015e (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 誌謝 I
Abstract II 中文摘要 III 目錄 i 圖目錄 iv 表目錄 v 第1章 緒論 1 1.1 腺苷概述 1 1.1.1 腺苷的形成與代謝 1 1.1.2 腺苷的運送 2 1.2 脂多糖與發炎反應 4 1.3 基因編輯技術之發展與應用 5 1.3.1 鋅指核酸酶 6 1.3.2 類轉錄激活因子效應物核酸酶 7 1.3.3 常間迴文重複序列叢集關聯蛋白 7 1.3.4 Cre-loxP系統在基因轉殖技術的應用 9 第2章 研究目的 14 第3章 實驗方法 15 3.1 實驗動物 15 3.2 基因剔除小鼠之產製 15 3.2.1 全身性ENT2基因剔除小鼠 (C57BL/6-Slc29a2em1)產製策略 15 3.2.2 條件性ENT2基因剔除小鼠 (C57BL/6-Slc29a2em1(flox)) 產製策略 16 3.3 基因轉殖小鼠的配種策略 17 3.3.1 ENT2 全身性剔除小鼠 (C57BL/6-Slc29a2em1) 17 3.3.2 ENT1全身性剔除小鼠 (B6.129X1-Slc29a1tm1Msg/J) 18 3.4 基因轉殖小鼠之基因型檢測 18 3.4.1 Genomic DNA之萃取 19 3.4.2 以PCR方法鑑定小鼠基因型 20 3.5 LPS誘發神經發炎之小鼠模型建立 22 3.6 清醒小鼠腦部微透析測定及胞外腺苷分析 22 3.6.1 微透析探針體外回收率測試 25 3.6.2 以超效液相層析儀 (UPLC) 分析體外回收率樣品之方法 25 3.6.3 腦部定位及埋置探針導管 26 3.6.4 清醒小鼠腦部微透析 27 3.6.5 以高效液相層儀 (HPLC) 分析胞外腺苷濃度值 28 3.7 RNA萃取及其品質檢測並製備反轉錄DNA 29 3.7.1 RNA萃取 29 3.7.2 反轉錄製備互補DNA 30 3.7.3 即時定量聚合酶連鎖反應 30 3.8 免疫組織化學染色法 31 3.8.1 小鼠灌流及腦片製備 32 3.8.2 免疫組織化學染色 33 3.9 數據分析 33 第4章 實驗結果 41 4.1 ENT1及ENT2於各腦區mRNA表現量 41 4.2 ENT2基因全剔除小鼠表徵與其基因型鑑定 41 4.3 ENT1及ENT2基因剔除小鼠海馬迴之細胞腺苷濃度基礎值 42 4.4 ENT2基因全剔除小鼠之腺苷相關蛋白的mRNA表現量比較 42 4.5 以LPS誘發ENT2基因全剔除小鼠之腦部發炎程度比較 43 4.5.1 以免疫染色法測定microglia activation及astrogliosis 43 4.5.2 小鼠皮質與海馬迴部位細胞激素之mRNA表現量測定 44 4.6 經LPS誘導腦部發炎後胞外腺苷之濃度變化 45 4.7 LPS誘導之發炎反應對腺苷相關蛋白之mRNA表現量的影響 45 第5章 討論 57 第6章 結論 61 參考資料 62 附錄 66 1. ENT2基因剔除小鼠離乳後至實驗前之體重變化 66 2. C57BL/6-Slc29a2em1(flox)小鼠基因鑑定方法之確認 67 3. 小鼠標號辨識 69 指號 69 耳號 70 | |
dc.language.iso | zh-TW | |
dc.title | ENT2在調控脂多糖所誘發神經發炎之角色探討 | zh_TW |
dc.title | The role of ENT2 in the modulation of lipopolysaccharide-induced neuroinflammation | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳儀莊(Yijuang Chern),林淑華(Shu-Wha Lin) | |
dc.subject.keyword | 第二型核?轉運蛋白,神經發炎,腺?, | zh_TW |
dc.subject.keyword | equilibrative nucleoside transporter 2,neuroinflammation,adenosine, | en |
dc.relation.page | 70 | |
dc.identifier.doi | 10.6342/NTU201603665 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-02-13 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥學研究所 | zh_TW |
dc.date.embargo-lift | 2024-03-11 | - |
顯示於系所單位: | 藥學系 |
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