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系統識別號 U0007-1704200714542199
論文名稱(中文) 骨碎補之類黃鹼素對蝕骨細胞活性的影響
論文名稱(英文) Effect of Flavonoids Isolated from Drynaria fortunei on Osteoclasts Activity
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) 許智政
研究生(英文) Chih Cheng Hsu
學號 m102092018
學位類別 碩士
語文別 中文
口試日期
論文頁數 95頁
口試委員 指導教授-許秀蘊
指導教授-孫瑞昇
中文關鍵字 骨碎補  蝕骨細胞 
英文關鍵字 Drynaria fortunei  osteoclasts  osteoprotegerin 
學科別分類
中文摘要 骨碎補(Gusuibu)為水龍骨科(Polypodiaceae)槲蕨(Drynaria fortunei (Kunze) J. Sm.)之乾燥根莖,根據過去許多的文獻記載,骨碎補為具有預防及治療骨相關疾病的傳統中藥。而根據本實驗室逐年建立之研究模式,骨碎補經活性分析證實對骨母細胞有促進之作用,而其分離之成分(-)-epicatechin-3-O-β-D-allopyranoside,也已於本實驗室證明對骨母細胞,有促進增生的作用 (簡尚志,2002)。本實驗再對骨碎補於蝕骨細胞,與骨質的增生和再吸收作用過程,對其活性機轉作更深入之探討。 本實驗用Wistar新生老鼠做細胞初代培養,以體外試驗模式探討骨碎補對蝕骨細胞的影響,經由不同時間點的細胞存活率測試(MTT assay),分析酸性磷酸酵素(ACP)和乳酸去氫酵素(LDH)的含量來測量酵素活性。並佐以酒石酸磷酸酵素染色(TRAP stain)觀察蝕骨細胞分化成熟過程的影響;用2'',7''-Dichlorofluorescein(DCF assay)測試蝕骨細胞內活性氧物種;抽取細胞DNA以電泳分析,研究蝕骨細胞凋亡(apoptosis)的表現。另外抽取蝕骨細胞中RNA,以同步定量聚合酶連鎖反應方法(real-time PCR),以TaqMan® probe檢視蝕骨細胞osteoprotegerin(OPG)和RANK-ligand(RANKL)的基因表現,是否具抑制蝕骨細胞活性的作用。 本實驗將從槲蕨根莖經純化分離出之(-)-epicatechin-3-O-β-D-allopyranoside,已於本實驗室證明對骨母細胞有促進增生的作用。就蝕骨細胞活性測試(MTT assay)而言,當加入(-)-epicatechin-3-O-β-D-allopyranoside (10 μg/mL)培養第七天時即呈現明顯的細胞抑制(p<0.05),且明顯抑制酸性磷酸酵素活性,這情形也表現在抗酒石酸磷酸酵素染色上。從細胞內的氧化壓力分析,(-)-epicatechin-3-O-β-D-allopyranoside促使活性氧物種濃度上升10%,而在細胞apoptosis表現上,實驗組和控制組間,發現並未導致蝕骨細胞有apoptosis的趨向。在RNA表現的方面,(-)-epicatechin-3-O-β-D-allopyranoside讓OPG表現增加並抑制了RANKL,所以有抑制蝕骨細胞的活性。 由以上的結果可以顯示(-)-epicatechin-3-O-β-D-allopyranoside對於骨質之再吸收有抑制的效果,其在抑制蝕骨細胞成熟和分化,並且降低成熟蝕骨細胞中酸性磷酸酵素的活性,而非經由細胞凋亡的機轉。
英文摘要 The traditional Chinese medicine: Gusuibu [Drynaria fortunei (Kunze) J. Sm.] (Polypodiaceae) was commonly used to manage disorders of orthopedics and has been claimed to have therapeutic effects on bone healing. In the preliminary study, the Gusuibu and its isolated pure compound: (-)-epicatechin-3-O-β-D-allopyranoside has been elucidated to be capable to enhance the proliferation of osteoblasts. In this study, we tried to further investigate the molecular mechanism of gusuibu on the bone cells metabolism. Primary culture of osteoclasts cells were isolated from newborn Wistar rats. The effects of Drynaria fortunei on bone cell viability were determined by the method of MTT assay. The effects on osteoclasts cells activities were analyzed by acid phosphatase (ACP) and lactate dehydrogenase (LDH). The differentiation of osteoclasts was analyzed by tartrate-resistant acid phosphatase stain. The quantification of intracellular reactive oxygen species (ROS) was assayed by 2'',7''-Dichlorofluorescein (DCF) method. Moreover, the RNA extracted from osteoclasts cells were applied on the real-time PCR, and then determined by TagMan® probe. The gene expression of OPG (osteoprotegerin) and RANKL (RANK-ligand) could further tell if they have negative influence on activity of osteoclast cells. The results showed that the (-)-epicatechin-3-O-β-D-allopyranoside [extracted from the rhizoma of Drynaria fortunei (KUNTZE) J. SMITH (Polypodiaceae)] has an adjuvant on proliferation of osteoblast cells. The 10 μg/mL (-)-epicatechin-3-O-β-D-allopyranoside inhibits the activity of osteoclasts during proliferation stage as manifested in the MTT assay, enzyme activity of ACP and TRAP stain. In the presence of (-)-epicatechin-3-O-β-D-allopyranoside, the intracellular reactive oxygen species level of osteoclasts increased. On the concept of RNA expression, (-)-epicatechin-3-O-β-D-allopyranoside improved the expression of OPG but meanwhile inhibited RANKL. It showed the inhibition of osteoclasts cells activity. The potential of (-)-epicatechin-3-O-β-D-allopyranoside on bone resorption was down-regulator on osteoclastic activity and inhibited the acid phosphatase activities but not induced the apoptosis of osteoclasts.
論文目次 目錄 中文摘要…………………………………………………………………VII 英文摘要…………………………………………………………………IX 第一章 緒論 1-1. 前言…………………………………………………………………1 1-2. 研究背景……………………………………………………………3 1-3. 研究目的……………………………………………………………5 第二章 理論基礎 2-1. 骨碎補(Drynaria fortunei(KUNTZE)J.SMITH)之藥理作用…6 2-1-1. 傳統中藥骨碎補之藥理……………………………………………6 2-1-2. 骨碎補近代之藥理…………………………………………………6 2-2. 骨科學理論基礎………………………………………………………7 2-2-1. 骨組織的結構………………………………………………………7 2-2-2. 骨的成分(Compositions of bone)……………………………8 2-2-3. 骨修復作用(Bone repairing)…………………………………9 2-2-4. 骨重塑作用(Bone remodeling)………………………………13 2-2-5. 內分泌對骨代謝的影響……………………………………………15 2-2-6. 骨質流失的預防……………………………………………………18 2-3. 骨骼細胞活性…………………………………………………………21 2-3-1. 骨細胞 (Osteocyte) 之生理活性………………………………21 2-3-2. 骨母細胞之生理活性………………………………………………21 2-3-3. 骨母細胞之特定蛋白表現因子……………………………………23 2-3-4. 蝕骨細胞之生理活性………………………………………………26 2-3-5. 蝕骨細胞和骨質疏鬆………………………………………………29 2-3-6. 蝕骨細胞分化過程中可定性定量的生化指標……………………31 2-3-7. 生物中活性含氧物種(reactive oxygen species, ROS)……33 2-3-8. 活性含氧物種與蝕骨細胞…………………………………………35 2-4. 細胞基因表現…………………………………………………………35 2-4-1. 反轉錄聚合酶連鎖反應(RT-PCR)………………………………35 2-4-2. 同步定量聚合酶連鎖反應(real-time PCR)…………………36 第三章 材料與方法 3-1. 實驗器材………………………………………………………………40 3-1-1. 實驗儀器設備………………………………………………………40 3-1-2. 藥品試劑……………………………………………………………41 3-1-3. 耗材…………………………………………………………………44 3-2. 實驗方法………………………………………………………………45 3-2-1. 骨母細胞培養………………………………………………………45 3-2-1-1. 動物犧牲與頭蓋骨之取得………………………………………46 3-2-1-2. 骨母細胞溶離與初代細胞培養…………………………………46 3-2-1-3. 分盤………………………………………………………………47 3-2-2. 蝕骨細胞培養………………………………………………………48 3-2-2-1. 肺藏之取得與單核球之沖離……………………………………48 3-2-1-2. 單核球與骨母細胞之共同培養…………………………………49 3-2-3. 中藥檢品之製備……………………………………………………51 3-2-3-1. 藥材之萃取………………………………………………………51 3-2-3-2. TLC展開液………………………………………………………52 3-2-3-3. 濃度換算…………………………………………………………52 3-2-4. 細胞活性分析………………………………………………………52 3-2-5. 酵素分析……………………………………………………………53 3-2-5-1. 乳酸脫氫酵素 (LDH) 分析……………………………………53 3-2-5-2. 酸性磷酸酵素 (ACP) 分析……………………………………56 3-2-6. 蝕骨細胞定性分析TRAP stain……………………………………57 3-2-7. 細胞氧化活性的測試………………………………………………58 3-2-8. DNA裂解梯度片段萃取……………………………………………61 3-2-9. 反轉錄聚合酶連鎖反應 (RT-PCR)………………………………62 第四章 結果與討論 4-1. 細胞活性測試結果( MTT Assay)……………………………………66 4-2. 細胞內外乳酸脫氫酵素 (LDH) 測試結果…………………………72 4-3. 細胞內外酸性磷酸酵素 (ACP) 測試結果…………………………76 4-4. 抗酒石磷酸酵素染色 (TRAP Stain) 結果…………………………78 4-5. 細胞氧化活性的測試…………………………………………………80 4-6. 細胞凋亡測試結果……………………………………………………82 4-7. 蝕骨細胞基因表現……………………………………………………83 第五章 總結與未來展望 第六章 參考文獻 表目錄 表3-1. 細胞培養計數標準及培養液體積…………………………………50 表4-1. 骨碎補初萃物對蝕骨細胞存活率的影響…………………………67 表4-2. (-)-epicatechin-3-O-β-D-allopyranoside對蝕骨細胞存活率的影響…………………………………………………………………………69 表4-3. (-)-epicatechin-3-O-β-D-allopyranoside(D)和17β-Estradiol(E2)對蝕骨細胞存活率的影響………………………………71 表4-4. (-)-epicatechin-3-O-β-D-allopyranoside 10 μg/mL對蝕骨細胞內外之LDH的影響…………………………………………………………73 表4-5. (-)-epicatechin-3-O-β-D-allopyranoside 100 μg/mL對蝕骨細胞內外之LDH的影響…………………………………………………………75 表4-6. (-)-epicatechin-3-O-β-D-allopyranoside 10 μg/mL對蝕骨細胞內外之ACP的影響…………………………………………………………77 表4-7. (-)-epicatechin-3-O-β-D-allopyranoside對蝕骨細胞DCF影響……………………………………………………………………………81 表4-8. 蝕骨細胞的TaqMan® probe基因表現……………………………84 圖目錄 圖2-1. 骨骼之解剖構造……………………………………………………8 圖2-2. 骨修復過程中骨原細胞、骨細胞與骨母細胞間的分化與調節關係……………………………………………………………………………11 圖2-3. 骨修復過程中新生骨組織的階段性替換情形……………………13 圖2-4. 骨重塑作用之動態平衡圖…………………………………………15 圖2-5. 影響骨骼平衡和骨密度的因子……………………………………16 圖2-6. 間葉細胞分化之細胞群系圖………………………………………22 圖2-7. 骨母細胞表型的發展 (in vitro development of the osteoblast phenotype)……………………………………………………25 圖2-8. 骨母細胞成熟分化過程及特定蛋白質表現………………………25 圖2-9. 蝕骨細胞分化過程中所需的cytokines和不同階段的生化指標……………………………………………………………………………27 圖2-10. 蝕骨細胞生成過程………………………………………………28 圖2-11. 雌激素對骨母細胞和蝕骨細胞的調節作用……………………30 圖2-12. 蝕骨細胞再吸收時釋放出type 5b Tartrate-Resistant Acid Phosphatase…………………………………………………………………32 圖2-13. 活性氧物種的形成方式和對抗活性氧化物危害的防禦機制…34 圖2-14. Real-time PCR螢光偵測方式……………………………………37 圖2-15. Real-time PCR反應所生成的螢光………………………………38 圖2-16. DNA聚合和SYBR® Green I產生螢光信號………………………38 圖2-17. DNA聚合和TaqMan® probe產生螢光信號………………………39 圖3-1 骨母細胞及蝕骨細胞培養流程圖…………………………………50 圖3-2. NADH與NAD之光譜吸收峰…………………………………………55 圖3-3. DCFH-DA與細胞內ROS產生螢光之過程……………………………59 圖3-4. (-)-epicatechin和Cu(II)氧化還原作用的路徑………………60 圖4-1. 骨碎補初萃物對蝕骨細胞存活率的影響…………………………67 圖4-2. (-)-epicatechin-3-O-β-D-allopyranoside對蝕骨細胞存活率的影響…………………………………………………………………………69 圖4-3. (-)-epicatechin-3-O-β-D-allopyranoside和17β-Estradiol對蝕骨細胞存活率的影響……………………………………………………71 圖4-4. (-)-epicatechin-3-O-β-D-allopyranoside 10 μg/mL對蝕骨細胞內外之LDH的影響…………………………………………………………73 圖4-5. (-)-epicatechin-3-O-β-D-allopyranoside 100 μg/mL對蝕骨細胞內外之LDH的影響…………………………………………………………75 圖4-6. (-)-epicatechin-3-O-β-D-allopyranoside 10 μg/mL對蝕骨細胞內外之ACP的影響…………………………………………………………77 圖4-7. (-)-epicatechin-3-O-β-D-allopyranoside 之TRAP染色結果……………………………………………………………………………79 圖4-8. (-)-epicatechin-3-O-β-D-allopyranoside對蝕骨細胞DCF影響……………………………………………………………………………81 圖4-9. 蝕骨細胞DNA fragmentation表現………………………………82
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