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研究生:謝承志
研究生(外文):HSIEH, CHENG-CHIH
論文名稱:Herbacetin於退化性關節炎細胞模型的影響
論文名稱(外文):Effects of Herbacetin on chondrocyte and synovial fibroblasts with IL-1β stimulation
指導教授:彭奕仁
指導教授(外文):PENG,YI-JEN
口試委員:鄭珈毘夏堪臺
口試委員(外文):CHENG,CHIA-PIHSIA,KAN-TAI
口試日期:2021-05-11
學位類別:碩士
校院名稱:國防醫學院
系所名稱:病理及寄生蟲學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:79
中文關鍵詞:退化性關節炎軟骨細胞滑膜成纖維細胞Herbacetin氧化壓力蛋白聚醣細胞外基質
外文關鍵詞:osteoarthritischondrocytesynovial fibroblastsHerbacetinoxidative stressglycosaminoglycanextracellular matrix
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退化性關節炎(Osteoarthritis, OA)是世界上常見的退化性疾病之一,有許多危險因子參與其中,包括年齡、肥胖、創傷等。在退化性關節炎中主要為軟骨代謝不平衡、產生發炎反應及氧化壓力,促使關節表面軟骨遭到破壞。滑膜組織在OA的發展上也佔有一席之地,在OA的變化中滑膜會不正常增生,使免疫細胞產生細胞激素,如IL-1β、TNF-α等,其中IL-1β的產生對於OA是至關重要的。目前在治療OA主要是以NSAID類藥物為主,但因其會對於腸胃道造成負擔,應此探討是否可以使用其他化合物作為OA治療的替代藥物,Herbacetin為一種活性黃酮類化合物,具有抗發炎、抗氧化和抗癌症的效果。經實驗發現Herbacetin對於IL-1β刺激軟骨及滑膜細胞後所產生的iNOS、MMPs及氧化壓力具有抑制效果,並對軟骨細胞誘導Nrf2進入細胞核中產生抗氧化蛋白質HO-1與NQO1。此外並會抑制軟骨細胞中Stat1路徑減少氧化壓力的產生。在體外軟骨培養中,利用Safrnin O染色及DMMB assay 發現Herbacetin可有效抑制GAG的降解。
綜合上述實驗結果,Herbacetin在對於減少氧化壓力及基質分解是有效的,但對於臨床藥物的使用,仍需再進行更進一步的研究。

Osteoarthritis (OA) is a common degenerative disease in the world, many risk factors involve the disease, including age, obesity trauma etc. Osteoarthritis result f imbalance of cartilage metabolism, inflammation and oxidative stress, which result in the destruction of joint cartilage. The synovium also plays a role in the progression osteoarthritis. In the osteoarthritis process, the synovium reveals mild hyperplasia and immune cells infiltration to produce cytokines. The IL-1β is a potent cytokine in osteoarthritic pathological progress. The current pharmacotherapy of OA is pain relief. However, on non-steroidal anti-inflammatory drugs, may cause a burden on the gastrointestinal tract and coagulopathy, Hence, we investigate the alternation compound.
Herbacetin is an active flavonoid that has anti-inflammatory, anti-oxidant and anti-cancer activities. We find the Herbacetin has an attenuating effects of iNOS, MMPs and oxidative stress on chondrocytes and synovial fibroblasts stimulated by IL-1β. The mechanism may be associated activation of Nrf2/HO-1 pathway. In addition, it inhibits the STAT1 pathway to reduce oxidative stress in chondrocyte. In ex vivo study, the Safarnin O staining and DMMB assay demonstrate that Herbacetin can effectively inhibit the degradation of extracellular matrix.
Based on the experimental results, Herbacetin may be value of in treatment of OA though its anti-oxidative and anti-degradative properties.

目錄
謝誌 I
中文摘要 II
英文摘要 IV
圖目錄 IX
第一章 緒論 1
1.1退化性關節炎(osteoarthritis) 1
1.1.1 退化性關節炎介紹 1
1.1.2 退化性關節炎的危險因子 3
1.1.3退化性關節炎與氧化壓力影響 4
1.1.4退化性關節炎中基質金屬蛋白酶的影響 5
1.1.5 滑膜組織對退化性關節炎的影響 7
1.1.6 退化性關節炎的治療 8
1.2 Herbacetin 10
1.2.1 Herbacetin來源及特性 10
1.2.2 Herbacetin抗發炎作用 10
1.2.3 Herbacetin的抗腫瘤能力 11
1.2.4 Herbacetin抗高血糖與高血脂作用 13
1.3 研究動機 13
1.4 研究目的 14
第二章 材料與方法 15
2.1 實驗材料 15
2.1.1 化學藥品 15
2.1.2 抗體 16
2.1.3引子 17
2.2實驗方法 18
2.2.1實驗設計流程圖 18
2.2.2 初級軟骨細胞與滑膜細胞培養 (Primary cultures) 19
2.2.3 細胞毒殺性試驗 (MTT assay) 22
2.2.4 蛋白質萃取及定量(Protein extraction & concentration determination) 23
2.2.5 西方墨點法 (Western blotting) 25
2.2.6 明膠酵素活性分析 (Gelatin Zymography) 28
2.2.7 活性氮化物濃度測定分析 (Griess reaction) 30
2.2.8 Reactive Oxygen Species 測定 (ROS detection) 31
2.2.9 電泳位移分析(Electrophoretic mobility shift assay ; EMSA) 32
2.2.10 軟骨體外培養實驗(Ex vivo) 33
2.2.11 石蠟切片與蘇木紫-伊紅染色法(H&E stain) 34
2.2.12 番紅-固綠染色(Safranin O-fast green stain) 35
2.2.13 二甲基亞甲基藍試驗(Dimethylmethylene blue assay, DMMB) 36
2.2.14 統計學分析(Statistical analysis) 37
第三章 結果 38
3.1 Herbacetin對於人類軟骨細胞與滑膜細胞的影響 38
3.1.1 Herbacetin對於人類軟骨細胞與滑膜細胞的生長影響 38
3.1.2 Herbacetin對於IL-1β刺激下人類軟骨細胞與滑膜細胞發炎物質的影響 38
3.1.3 Herbacetin對於IL-1β刺激下人類軟骨細胞與滑膜細胞的活性氮化物產生影響 39
3.1.4 Herbacetin對於IL-1β刺激下人類軟骨細胞與人類滑膜細胞ROS產生影響 40
3.1.5 Herbacetin對於IL-1β刺激下人類軟骨細胞與人類滑膜細胞分泌MMPs產生影響 40
3.1.6 以Zymography分析Herbacetin對於IL-1β刺激下人類軟骨細胞與滑膜細胞分泌MMP-2、9產生影響 41
3.1.7 Herbacetin對於IL-1β刺激下人類軟骨細胞與人類滑膜細胞誘導NRF2產生HO-1與NQO1的影響 42
3.1.8 Herbacertin對於人類軟骨細胞與滑膜細胞的訊息傳遞調控 43
3.2 Herbacetin對於體外培養軟骨模型下ECM的變化 44
第四章 討論 45
4.1Herbacetin對於IL-1β誘導人類軟骨細胞與滑膜細胞的影響 45
第五章 結論 49
第六章 參考文獻(References) 50
附圖 54


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