系統識別號 U0007-1704200714553830
論文名稱(中文) 3-D培養模式下脫氫表雄酮對軟骨細胞的影響
論文名稱(英文) Effects of dehydroepiandrosterone on chondrocytes in a three-dimensional culture model
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 93
學期 2
出版年 94
研究生(中文) 吳俊賢
研究生(英文) Chun-Xian Wu
學號 M301092003
學位類別 碩士
語文別 中文
論文頁數 87頁
口試委員 指導教授-許秀蘊
中文關鍵字 軟骨細胞  三度空間培養法  脫氫表雄酮  脂多醣 
英文關鍵字 chondrocytes  3-dimensional cultures  Dehydroepiandrosterone  Lipopolysaccharide 
中文摘要 摘 要 隨著年紀的增加、生理性關節活動變小或重力的壓迫,例如肥胖會使軟骨退化、磨損、關節表面凹凸不平,嚴重時軟骨會完全喪失,骨頭與骨頭直接接觸會導致發炎、紅腫、疼痛,甚至無法正常彎曲、移動。由於身體的老化過程,不能再生產足夠的蛋白多醣和合成膠質來維持健康的軟骨結構,老化的關節愈來愈沒有彈性,而造成關節軟骨的磨損。脫氫表雄酮Dehydroepiandrosterone (DHEA)是一種人體重要的荷爾蒙,DHEA是性荷爾蒙的前驅物,由腦部、皮膚和腎上腺所製造,也可由山藥、芋類植物或鹿茸中萃取得到,在體內會轉變成為雄性素與雌性素,這兩種激素在人體中對於骨的維持非常地重要,所以可能因此而對軟骨有幫助。本研究欲探討在軟骨受傷害的情況下,是否有治療或保護的作用,更進一步的說,或許可提供另一個對於軟骨傷害治療的新方向。 本實驗取用初生四天之幼鼠為動物模式,取其膝關節軟骨培養軟骨母細胞,並採用三度空間培養法 (3-dimensional cultures) 培養軟骨細胞,然後在加入Dehydroepiandrosterone (DHEA) 或在LPS與SNAP分別來模擬細菌性關節炎與自然老化退化性關節炎的情形,並檢測軟骨細胞增生情形,再進一步的檢測軟骨細胞內基質glycosaminoglycans (GAG),使用DMB assay,而total collagen量的多少,則使用OHP assay及一些會造成軟骨傷害的物質的表現,如nitric oxide (NO)、prostgalends (PGE2)、Interleukin-6 (IL-6)、metalloproteinases (MMPs)。並且會探討MMPs與TIMP-1的表現,與即時定量反轉錄聚合?連鎖反應來探討基因COX-2、iNOS、MMP1、MMP3、MMP13。 實驗結果顯示,DHEA在誘導軟骨細胞增生時,在10-6M濃度下時最好,在加有H2O2 情況時,在10-6M濃度下DHEA對自由基H2O2的傷害有保護作用,LPS可以誘導NO、PGE2、IL-6、MMP1、MMP3、MMP13產生,但可以抑制GAG、total collagen、TIMP-1合成。DHEA可以部分的減少NO、PGE2、MMP1、MMP3、MMP13產生,但對IL-6則沒有影響。所以DHEA在軟骨細胞受傷害的情況下具有保護作用。我們將DHEA的濃度提高到10-5M,對軟骨細胞並沒有毒性傷害,而且還可調節MMP 與TIMP-1之間平衡,所以DHEA可扮演一個對抗軟骨傷害的角色。
英文摘要 Abstract With increasing age, the activity of joint becomes less or pressed by gravity. For example, fatness will make cartilage atrophy, bruised and surfaces of joint will not be even, more seriously, cartilage will completely lose the function. Direct contacts among bones will lead to infection, inflammation, pains and even abnormal curves and motions. Due to aging process of body, it is impossible to generate enough proteoglycan and collagen in order to keep the structure of cartilage healthy. Thus, aging joints become less elastic, causing the bruise of joints of cartilage. Dehydroepiandrosterone (DHEA) is an important hormone of human body which is the precursor of sexual hormone, and made from brain, skin and adrenal glands. It can also be made from Dioscorea、Taro plant or young antlers. It can be transformed into male and female hormones that are very important to the maintenance of the bone in the body. This research aims to study whether it can heal or protect chondrocytes under harmful situation or offer an alternative new treatment for cartilage damage. Chondrocytes were isolated from mouse knee cartilage and cultured three-dimensionally in agarose. Then simulating the proliferation of infectious arthritis and osteoarthritis by adding DHEA with LPS, or DHEA with SNAP, GAG were measured with DMB assay, total collagen with OHP assay, (NO、PGE2、IL-6、MMPs) were measured with specific kits respectively. Furthermore, the cells transcriptional expressed the detectable levels of COX-2、iNOS、MMP1、MMP3 and MMP13 mRNAs. This experiment showed revealed that DHEA induced an increasing cell proliferation with the optimum effects at 10-6M. LPS increased the production of NO、 PGE2、IL-6、MMP1、MMP3 and MMP13, but inhibited the synthesis of GAG、OHP and TIMP-1. DHEA partially revered the effect of LPS on NO、PGE2、MMP1、MMP3 and MMP13 production, had no effect on IL-6 production. So DHEA has the function of protection to the newborn mice chondrocytes under the condition of being harmed. Our study demonstrated DHEA has no toxic effect on chondrocytes up to 10-5M of concentration and has an ability to modulate the imbalance between MMPs and TIMP-1, which suggest that it has a protective role against articular cartilage loss.
論文目次 中文摘要 I 英文摘要 II 目錄 III 圖目錄 VII 表目錄 X 第一章 緒論 1 1 1-1 前言 1 1-2 研究目的 3 第二章 理論基礎 4 2-1骨骼的發生的兩種方式 4 2-1-1 膜性骨的形成方式 4 2-1-2 軟骨性骨的形成方式 4 2-2軟骨細胞 (chondrocytes) 6 2-2-1 膠原蛋白(collagen) 7 2-2-2 醣蛋白(proteoglycan) 8 2-2-3 Matrix metalloproteinase ( MMPs ) 9 2-2-4 Nitric oxide synthases(NOS) 12 2-2-5 前列腺素(prostaglandins) 15 2-2-6 氧化性傷害 17 2-2-7 Dehydroepiandrosterone (DHEA) 19 2-2-8 造成軟骨損傷原因 20 2-2-9 骨關節炎(Osteoarthritis) 21 2-2-10 治療方法2-2-11 軟骨移植 2224 第三章 材料與方法 25 3-1 材料 25 3-1-1 實驗儀器 25 3-1-2 實驗試藥與耗材 26 3-2 方法 29 3-2-1 試藥配製 29 3-2-1-1 細胞培養試藥配製 29 3-2-1-2 Agarose cell specimens溶解試藥配製 31 3-2-2 三度空間培養方法 32 3-2-2-1 軟骨細胞的培養方法 32 3-2-2-2 Agarose cell specimens溶解方法 33 3-2-3 細胞活性分析 39 3-2-3-1 反應試藥的準備 39 3-2-3-2 分析步驟 39 3-2-4 Dimethylmethylene blue assay (GAG) 40 3-2-4-1 反應試藥的準備 40 3-2-4-2 分析步驟 41 3-2-5 Griess reaction (NO) 41 3-2-5-1 反應試藥準備 41 3-2-5-2 分析步驟 42 3-2-6 Hydroxyproline assay (OHP) 42 3-2-6-1 反應試藥準備 42 3-2-6-2 分析步驟 43 3-2-7 Prostaglandin E2 immunoassay 44 3-2-7-1 反應試藥準備 44 3-2-7-2 分析步驟 44 3-2-8 Tissue inhibitor of Metalloproteinases immunoassay (TIMP-1) 45 3-2-8-1 反應試藥準備 45 3-2-8-2 分析步驟 45 3-2-9 Interleukine-6 immunoassay (IL-6) 47 3-2-9-1 反應試藥準備 47 3-2-9-2 分析步驟 47 3-2-10 即時定量反轉錄聚合?連鎖反應 49 3-2-10-1 Total RNA之純化 49 3-2-10-1-1 前處理 49 3-2-10-1-2 Total RNA isolation 50 3-2-10-1-3 Total RNA含量分析 50 3-2-10-2 反轉錄合成cDNA 50 3-2-10-3 即時定量聚合?連鎖反應 51 第四章 結果與討論 53 4-1 MTT assay 53 4-1-1細胞活性與細胞毒性分析 53 4-1-2 Dehydroepiandrosterone (DHEA)對軟骨細胞增生的影響 54 4-1-3 H2O2存在的情況下DHEA對軟骨細胞的影響 55 4-2 細菌感染之細菌性關節炎 57 4-2-1 LPS存在的情況下對軟骨細胞的影響 58 4-2-2 DHEA對LPS誘導軟骨細胞產生PGE2的影響 59 4-2-3 10-6M DHEA對COX-2 mRNA的表現 60 4-2-4 DHEA對LPS誘導軟骨細胞產生NO的影響 61 4-2-5 10-6M DHEA對iNOS mRNA的表現 62 4-2-6 DHEA對LPS誘導軟骨細胞產生IL-6的影響 65 4-2-7 DHEA對MMP mRNA的表現 4-2-7-1 DHEA對MMP1 mRNA的表現 4-2-7-2 DHEA對MMP3 mRNA的表現 4-2-7-3 DHEA對MMP13 mRNA的表現 67676768 4-2-8 DHEA對LPS抑制軟骨細胞產生TIMP-1的影響 69 4-2-9 DHEA對LPS抑制軟骨細胞產生GAG的影響 71 4-2-10 DHEA對LPS抑制軟骨細胞產生total collagen的影響 72 4-3自然老化之退化性關節炎 74 4-3-1 DHEA抑制軟骨細胞產生NO的影響 74 4-3-2 DHEA對SNAP提供軟骨細胞NO的影響 76 4-3-3 DHEA對SNAP提供軟骨細胞NO抑制GAG的影響 77 第五章 總結與未來展望 81 參考文獻 82
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