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研究生:林育如
研究生(外文):Yu-Ju Lin
論文名稱:探討低氧誘導因子以及葡萄糖轉移子對於葡萄糖胺治療退化性關節炎中的分子機制(一)
論文名稱(外文):Study HIF-1α and GLUT-1 molecular mechanisms of glucosamine HCl on osteoarthritis (一)
指導教授:陳建和陳建和引用關係
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗生物技術學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:71
中文關鍵詞:退化性關節炎細胞激素細胞間質水解酵素軟骨瘤細胞葡萄糖胺低氧誘導因子葡萄糖轉移子
外文關鍵詞:osteoarthritisIL-1βmatrix metalloproteinaseschondrosarcoma cellglucosamine HClhypoxia-induced factor-1αglucose transporter-1
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  • 收藏至我的研究室書目清單書目收藏:1
依據臨床發現,退化性關節炎病人口服葡萄糖胺 (glucosamine HCl) 最後到達血液濃度並不如in vitro實驗研究的有效濃度,卻仍對病人的疼痛及關節修復有一定的效果,因此本實驗使用葡萄糖胺glucosamine HCl來觀察其對於IL-1β誘發chondrosarcoma SW1353細胞間質水解酵素 (Matrix Metallo- proteinase)的影響,發現高劑量的glucosamine HCl可以抑制低濃度IL-1β所引起的MMPs的mRNA及蛋白質表現,也可以抑制IL-8的mRNA表現,並且使用低劑量的glucosamine HCl則可以抑制高濃度IL-1β引起的發炎反應,實驗結果與臨床應用glucosmaine HCl治療關節炎的現象相符。另外,為了更接近關節環境,本實驗另進行正常氧氣濃度與低氧環境的對照,我們發現除了低氧環境會誘導低氧誘導因子 (HIF-1α) 蛋白表現之外,在低氧環境下單純使用IL-1β誘導的MMPs蛋白表現會降低,而且glucosamine HCl抑制MMPs的效果也會更明顯,同時我們也發現軟骨細胞受到IL-1β及低氧環境的誘導,葡萄糖轉移子 (GLUT-1) 表現量上升,依上述結果推論,HIF-1α及GLUT-1可能是glucosamine治療退化性關節炎的重要機制。
Clinical studies have shown that after oral administration of a therapeutic dose of glucosamine HCl, a low blood concentration of glucosamine can be achieved, but it does work to diminish pain and repair the cartilage for osteoarthritic patients. Although in vitro data cannot be extrapolated to pathophysiological conditions in humans, our data suggest the effects of action by which glucosamine HCl could modulate IL-1β induced MMPs expression by chondrosarcoma SW1353 cells. The data shows high concentration of glucosamine HCl can reduce the mRNA and protein expression of MMPs which induced by low concentration of IL-1β; low concentration of glucosamine HCl can reduced the inflammatory response up-regulated by high concentration of IL-1β. The data is to comform to clinical application. Besides, we compare the different effects under normoxia and hypoxia to draw on the real condition in human cartilage. HIF-1α protein level would be up-regulated under hypoxia, and furthermore, the level of MMPs protein expression that induced by IL-1β was lower under hypoxia condition. The inhibition of MMPs by glucosamine HCl was more in apparent. We also found GLUT-1 protein expression would up-regulated under IL-1β and hypoxia treatment by chondrocytes. To sum up the results, we suggested HIF-1α and GLUT-1 may be the important mechanisms for therapeutic of glucosamine HCl on osteoarthritis.
目錄
誌謝 I
摘要 II
Abstract IV
目錄 VI
圖目錄 VIII
第一章 緒論 1
第二章 文獻回顧 4
一.關節腔 4
(一)軟骨細胞 (chondrocyte): 4
(二)細胞外間質 (Extracellular Matrix) 4
二.退化性關節炎Osteoarthritis (OA): 5
三. 軟骨關節氧氣微環境以及在微量氧氣下的生存因子 (survival factor) →Hypoxia-inducible factor 1 (HIF-1): 7
四. 葡萄醣胺 (glucosamine): 9
五. 細胞激素 (cytokine): 11
六. 間質分解酵素 (matrix metalloproteinases; MMPs): 12
七.趨化激素 (chemokine): 13
八.葡萄糖轉移子-1 (Glucose transportor-1): 15
第三章 實驗儀器與器材 17
一.實驗細胞株: 17
二.藥劑 17
三.常用溶液 19
第四章 實驗方法 20
一.人類軟骨瘤細胞培養: 20
二.WST-1 Cell Proliferation Assay System 20
三.細胞RNA萃取 (Preparation of cell RNA) 21
四.RNA定量法 (RNA Assay) 21
五.反轉錄-聚合酶連鎖反應 (Reverse transcriptase-polymerase chain reaction,RT-PCR) 22
六.電泳 (electrophoresis) 24
七.細胞蛋白萃取 24
八.蛋白質定量法 (protein assay) 24
九.細胞培養液濃縮 25
十.西方墨點法 (Western bloting) 25
十一.統計分析 (Statistical analysis) 27
第五章 結果 28
一. 軟骨瘤細胞SW1353在IL-1β以及葡萄糖胺 (glucosamine)之刺激下並且在高低氧氣濃度中的生存力 28
二. 不同濃度的葡萄糖胺 (glucosamine)對於軟骨瘤細胞SW1353的保護作用以及高低濃度的IL-1β引起SW1353 MMP基因的表現 29
三. 葡萄糖胺 (glucosamine)會抑制經由IL-1β誘發的對軟骨瘤細胞SW1353代謝方面 (catabolic face)基因的影響 30
四. 葡萄糖胺 (glucosamine)會抑制經由IL-1β誘發的對軟骨瘤細胞SW1353合成方面 (anabolic face)基因的影響 31
五. 依據處理不同時間低氧環境 (hypoxia)或不同濃度的IL-1β可導致HIF-1α以及GLUT-1蛋白質表現量不同 31
六. 低氧環境 (O2 < 3 %)可以誘導軟骨瘤細胞SW1353 HIF-1α及GLUT-1蛋白表現量上升並且抑制間質水解酵素 (MMP-1, MMP-3)經由細胞激素IL-1β所誘發的蛋白質分泌量的表現 32
七. 比較在高低氧氣環境中,預先處理葡萄糖胺對於IL-1β所造成的HIF-1α, GLUT-1, MMP-1, 及MMP-3 誘導的影響 33
第六章 討論 35
第七章 參考文獻 44



圖目錄
Fig. 1 Effects of IL-1β, glucosamine, and hypoxia on cell viability in chondrosarcoma cells 49
Fig. 2 Effects of IL-1β and glucosamine on MMP-3 gene expression by chondrosarcoma cells 50
Fig. 3 Effects of high concentration of glucosamine on low concentration of IL-1β induced MMPs and IL-8 gene expression by chondrosarcoma cells. 52
Fig. 4 Effects of low concentration of glucosamine on high concentration of IL-1β induced MMPs and IL-8 gene expression by chondrosarcoma cells. 55
Fig. 5 Effects of high/low concentration of glucosamine on low/high concentration of IL-1β induced aggrecan gene expression by chondrosarcoma cells. 58
Fig. 6 HIF-1α and GLUT-1 protein expression was induced by IL-1β and hypoxia 60
Fig. 7 Effects of IL-1β on HIF-1α, GLUT-1, and MMPs protein expression under normoxia and hypoxia by chondrosarcoma cells 62
Fig. 8 Effects of high concentration of glucosamine on low concentration of IL-1β induced HIF-1α, GLUT-1, MMP-1, and MMP-3 protein expression under normoxia or hypoxia by chondrosarcoma cells 64
Fig. 9 Effects of low concentration of glucosamine on high concentration of IL-1β induced HIF-1α, GLUT-1, MMP-1, and MMP-3 protein expression under normoxia or hypoxia by chondrosarcoma cells 67

附圖 一 HIF-1α regulation by proline hydroxylation 70
附圖 二Glucosamine inhibit IL-1βinduced HIF-1α, GLUT-1, MMP-1, MMP-3, MMP-13, IL-8, and aggrecan expression by transport into chondrosarcoma cells via GLUT-1 71
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