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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 | 林能裕(Neng-Yu Lin) | |
dc.contributor.author | Hsiao-Han Hsu | en |
dc.contributor.author | 徐小涵 | zh_TW |
dc.date.accessioned | 2021-06-17T08:23:09Z | - |
dc.date.available | 2022-08-26 | |
dc.date.copyright | 2019-08-26 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74179 | - |
dc.description.abstract | 關節炎(Osteoarthritis)是一種具有多種風險因素的異質性疾病。其中,肥胖對承受重量和非承受重量之關節都有重大影響。在肥胖者之中由於承受重量過大使脂肪組織和軟骨細胞製造全身性發炎因子和關節炎的發生率有絕對的關係。有研究發現具有心臟代謝異常的肥胖患者的關節炎患病率和發病率高於健康的肥胖患者,這說明新的關節炎表型出現,即代謝綜合症引發的關節炎(Metabolic syndrome associated osteoarthritis),糖尿病患者亦包含在內 [1]。我們假設與代謝
因子異常的狀況包含葡萄糖過量導致毒性和肥胖組織製造促發炎因子TNF-α 對於軟骨前驅細胞所造成之影響。 O-linked β-linked N-acetylglucosamine(乙醯葡萄醣胺)為修飾細胞內蛋白質的醣類結構,簡稱為O-GlcNAc。目前發現受到兩種酵素專一性的調控胞內蛋白質的加醣與去醣作用(乙醯葡萄醣胺轉移酶(O-GlcNAc transferase)負責加醣;乙醯葡萄醣胺水解酶(O-GlcNAcase)負責去醣)。O-GlcNAcylation 作用於胞內蛋白的絲 胺酸(Serine)和蘇胺酸(Threonine)上氫氧基。O-GlcNAcylation 涉及細胞內眾多生理機制,失調後會引起許多疾病產生。本研究旨在探討關節炎中O-GlcNAcylation在關節炎中的角色。我們的研究發現,高糖刺激軟骨前驅細胞使胞內O-GlcNAc 表現提升,同時觀察到發炎反應增強,以藥物Thiamet G 抑制OGA 蛋白活性使胞內O-GlcNAcylation 蛋白累積後能夠改善軟骨前驅細胞發炎現象同時降低發炎因子MMP13 蛋白表現。敲落OGT 基因使O-GlcNAc 不能累積使發炎加劇。 綜合以上結論,我們的結果指出,透過藥物Thiamet G 促進細胞內O-GlcNAc修飾將有助於降低發炎程度。O-GlcNAcylation 是如何調控關節炎相關發炎基因的表現,並且降低發炎表現是我們未來的研究重點。 | zh_TW |
dc.description.abstract | Osteoarthritis (OA) is a heterogeneous disorder with several risk factors. Among them, obesity has a principal impact on both loading and non-loading joints. Mechanical overload and activity of systemic inflammatory mediators derived from chondrocyte or synoviocyte provide clues to the increased incidence and prevalence of OA in obesity. Recently, research found greater OA prevalence and incidence in obese patients with cardiometabolic disturbances than “healthy” obese patients, which led to the description of a new OA phenotype–metabolic syndrome (MetS)-associated OA. Certainly, individual metabolic factors including diabetes may increase the risk of obesity-induced OA. We hypothesized that abnormalities in metabolic factors with metabolic syndrome include excess glucose leading to the effects of toxic and obese tissue on the production of pro-inflammatory TNF-α on chondrocyte precursor cells.
O-GlcNAcylation is a dynamic modification of serine or threonine hydroxyl moieties nucleocytoplasmic proteins by UDP-GlcNAc. The dynamic and inducible cycling of the modification is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove the O-GlcNAc (O-linked β-linked N-acetylglucosamine) moiety respectively. The O-GlcNAcylation involes in many important cellular pathways and unbalance of O-GlcNAcylation has implicated in many kinds of diseases. O-GlcNAc glycosylation acts on intracellular proteins of urinium and sulphate. O-GlcNAcylation involves many physiological mechanisms in the cell, which can cause many diseases after dysregulation. This study was to investigate the association of O-GlcNAcylation with chondrocytes in osteoarthritis. Our study found that inhibition of OGA protein activity by the drug Thiamet G can improve the reduction of inflammation after accumulation of intracellular O-GlcNAcylation protein. For instance, high glucose and cellular inflammatory factors stimulate chondrocyte inflammatory response and cause intracellular O-GlcNAc expression rise. Elimination of OGT gene makes O-GlcNAc unable to accumulate and causes an increase in inflammatory response. Above all, our findings suggest that O-GlcNAcylation regulates inflammation phenotypes of ATDC5 cells through upregulating O-GlcNAc. These results regarding O-GlcNAcylation may also provide an alternative approach for osteoarthritis therapy by modulating O-GlcNAcylation modification. | en |
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dc.description.tableofcontents | 目錄(Contents)
誌謝 ........................................................................................................................... i 摘要 ......................................................................................................................... iii Abstract .................................................................................................................. iv 目錄(Contents) ....................................................................................................... vi 圖目錄(List of Figures) .......................................................................................... ix 表目錄(List of Tables) ............................................................................................ x 第一章 緒論(Introdction) .............................................................................. 11 1.1 關節炎(Osteoarthritis) .......................................................................... 11 1.1.1. 背景 ................................................................................................ 11 1.1.2. 關節炎的風險因子 ........................................................................ 11 1.1.3. 關節炎的分類與臨床分期 ............................................................ 14 1.1.4. 病理分類: .................................................................................... 15 1.1.5. 臨床分期: .................................................................................... 16 1.2 乙醯葡萄醣胺(O-GlcNAc) ........................................................................ 18 1.2.1. O-GlcNAc 在軟骨基質平衡中所扮演的角色 .............................. 18 1.2.2. O-GlcNAc 與關節炎的關係 .......................................................... 18 1.2.3. O-GlcNAcylation ............................................................................ 20 1.2.4. OGT 與OGA ................................................................................. 26 第二章 研究目的(Aim) .................................................................................. 28 2.1 探討O-GlcNAc 在關節炎臨床的重要性 ................................................. 28 2.2 調控O-GlcNAc 對於關節炎的影響 ......................................................... 28 第三章 研究方法與材料(Materials & Methods) ......................................... 29 vii 3.1 實驗材料(Materials) .................................................................................. 29 3.1.1. 抗體(Antibody) .............................................................................. 29 3.1.2. 引子(Primers) ................................................................................. 29 3.1.3. 其他(Others) ................................................................................... 29 3.2 免疫組織染色法(IHC) ......................................................................... 31 3.3 組織石蠟包埋與切片(Paraffin embedding & paraffin section) .......... 32 3.4 番紅-固綠染色(Safranin O&Fast Green stain) .................................... 33 3.5 細胞株及細胞培養(Cell line & Cell culture) ....................................... 33 3.6 藥物處理(Drug treatment) .................................................................... 33 3.7 細胞免疫螢光染色(ICC) ...................................................................... 34 3.8 蛋白質定量(Bradford protein assay) .................................................... 34 3.9 西方點墨法(Western blot) .................................................................... 35 3.10 RNA 萃取與RT-PCR(RNA Extraction & RT-PCR) ........................... 36 3.11 Real-time PCR ............................................................................................ 37 3.12 TEM 穿透式電子顯微鏡(Transmission Electron Microscopy) ........... 38 3.13 肥胖小鼠模型(High-fat diet induced osteoarthritis) ............................ 39 3.14 糖尿病小鼠模型(Streptozocin induced type 1 diabetes) ..................... 39 第四章 結果(Results) ..................................................................................... 40 4.1 HFD 小鼠與對照組小鼠膝關節組織完整度及O-GlcNAc 表現量 ........ 40 4.2 藥物streptozotocin 誘發第一型糖尿病小鼠合併症膝關節炎出現 ........ 40 4.3 以葡萄糖刺激軟骨前驅細胞株,O-GlcNAc 表現上升,導致發炎加 劇,給予Thiamet G 能降低發炎反應 ..................................................... 41 4.4 以TNF-α 刺激軟骨前驅細胞株,O-GlcNAc 表現下降,導致發炎加 劇,給予Thiamet G 專一性抑制OGA 蛋白活性,累積胞內O-GlcNAc 在蛋白質上修飾程度,發炎程度下降 .................................................... 43 4.5 Ogt 基因敲落(gene knockdown)加劇發炎程度 .................................. 44 4.6 TNF-α 改變胞內粒線體型態,給予Thiamet G 使粒線體型態趨於正常 45 viii 第五章 結論(Conclusion) .............................................................................. 46 第六章 討論(Discussion) ................................................................................ 47 6.1 O-GlcNAc 為細胞分化發育所必須 .......................................................... 47 6.2 葡萄糖在膝關節的角色 ............................................................................ 48 6.3 O-GlcNAc 在膝關節的角色 ...................................................................... 49 第七章 展望(Future work) ............................................................................ 51 7.1 研究受到O-GlcNAc 特定調控之蛋白 ..................................................... 51 7.2 研究O-GlcNAc 降低發炎之路徑 ............................................................. 51 圖表(Tables & Figures) ........................................................................................ 53 參考文獻(References) ........................................................................................... 63 ix 圖目錄(List of Figures) 圖 1:HFD 小鼠膝關節軟骨組織切片 .............................................................. 53 圖 2:STZ 誘發糖尿病小鼠膝關節軟骨組織 .................................................... 54 圖 3:HFD 小鼠膝關節軟骨組織免疫染色 ....................................................... 55 圖 4:STZ 小鼠膝關節軟骨組織免疫染色 ........................................................ 56 圖 5:葡萄糖誘使O-GlcNAc 上升 ..................................................................... 57 圖 6:不同時間點以Thiamet G 處理軟骨前驅細胞 ......................................... 58 圖 7:Thiamet G 藉由提高O-GlcNAc 以降低高糖之發炎反應 ...................... 59 圖 8:TNF-α 誘使ATDC5 之O-GlcNAc 下降。ATDC5 細胞螢光染色 ........ 60 圖 9:K.D. ogt 加劇MMP13 表現 ...................................................................... 61 圖 10:電子顯微鏡細胞切片 .............................................................................. 62 表目錄(List of Tables) 表 1:關節炎嚴重程度分期 ................................................................................ 17 表 2:Kellgren and Lawrence system 關節炎X 光造影分期差別 ..................... 18 | |
dc.language.iso | zh-TW | |
dc.title | 探討Thiamet G藥物增強的醣基化作用所調控TNF-α和高糖所誘導之軟骨細胞MMP13表達 | zh_TW |
dc.title | Augmented O-GlcNAcylation by Thiamet G downregulates TNF-α and High Glucose induced chondrocyte MMP13 expression | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鞏秀妮(kunghsiuni@gmail.com),王至弘(Jyh-Horng Wang) | |
dc.subject.keyword | 乙醯葡萄醣胺;乙醯葡萄醣胺轉移?;乙醯葡萄醣胺水解?;Thiamet G;腫瘤壞死因子-α;高糖;關節炎, | zh_TW |
dc.subject.keyword | O-GlcNAc,O-GlcNAc transferase,O-GlcNAcase,Thiamet G,TNF- α,high glucose,Osteoarthritis, | en |
dc.relation.page | 63 | |
dc.identifier.doi | 10.6342/NTU201903534 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-13 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 解剖學暨細胞生物學研究所 | zh_TW |
顯示於系所單位: | 解剖學暨細胞生物學科所 |
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