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研究生:張棋楨
研究生(外文):Chi-Ching Chang
論文名稱:含鈣晶體關節炎侵蝕骨頭(蝕骨細胞分化)的分子機制及表面增強拉曼光譜檢測含鈣晶體
論文名稱(外文):Molecular mechanisms of bone erosion (osteoclastogenesis) in calcium contained crystal–induced arthritis and Surface-enhanced Raman spectroscopy to detect CPPD crystals
指導教授:林時宜
指導教授(外文):Shyr-Yi Lin
口試委員:朱士傑李建和賴振宏
口試委員(外文):Shi-Jye ChuChian-Her LeeJenn-Huang Lai
口試日期:2014-12-22
學位類別:博士
校院名稱:臺北醫學大學
系所名稱:臨床醫學研究所
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:2016
畢業學年度:104
語文別:英文
論文頁數:77
中文關鍵詞:含鈣晶體蝕骨細胞分化表面增強拉曼光譜焦磷酸鈣晶體
外文關鍵詞:Calcium-containing crystalOsteoclastogenesisSurface-enhanced Raman spectroscopyCalcium pyrophosphate dehydrate crystal
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含鈣晶體性關節炎會引起骨頭被侵蝕造成關節破壞功能受損,從組織病理學報告可見到晶體與骨頭細胞的接觸。含鈣晶體性關節炎常見的有二種,第一種為假痛風其晶體為焦磷酸鈣鹽結晶(calcium pyrophosphate dihydrate ),第二種為基本磷酸鈣鹽結晶(basic calcium phosphate )沉澱造成之關節炎。蝕骨細胞是由單核細胞/巨噬細胞的前驅細胞在M-CSF 及RANKL存在下演化而來,成熟蝕骨細胞會表現出TRAP、Cathepsin-K 及vitronectin 受體。發炎性晶體性關節炎引起骨頭被侵蝕的分子機制是否會如同這些自體免疫發炎性關節炎經由RANK-RANKL-OPG 路徑而破壞骨頭,至今仍然不清楚。此外臨床上,含鈣晶體性關節炎病人的確診是抽取病人之關節液,再以偏光顯微鏡觀察,然而敏感度和準確率不夠高,若能發展以表面增強拉曼光譜敏感檢測含鈣晶體性關節炎的創新方法,可提高診斷含鈣晶體性關節炎病人的準確率,即早預防與治療。
本研究分兩部分進行,第一部分,含鈣晶體(焦磷酸鈣鹽結晶及基本磷酸鈣鹽結晶)會刺激蝕骨前驅細胞分化成熟,並導致骨頭被侵蝕,此研究之目的就是要闡明含鈣晶體性關節炎引起骨頭被侵蝕的分子機制。第二部分用表面增強拉曼光譜檢測含鈣晶體,此研究之目的是用創新方法去診斷含鈣晶體性關節炎。
第一部分的研究結果顯示,三種含鈣晶體(calcium-containing crystals-hydroxyaptite (HA),β-tricalcium phosphate (ß-TCP),and calcium pyrophosphate dehydrate (CPPD) 在M-CSF 及RANKL存在下會刺激蝕骨前細胞分化為成熟蝕骨細胞並會侵蝕骨頭,其證據為TRAP-positive 的單核細胞數目,TRAP 活性和骨片吸收面積均呈現dose dependent顯著增加,此外TRAP, cathepsin-K and CTR 的訊號RNA亦顯著表現, 三種含鈣晶體刺激蝕骨前細胞分化為成熟蝕骨細胞乃經由活化MAPKs 訊號傳導路徑。
第二部分的研究結果:以銀層沉積在金奈米粒子表面,製造高表面增強拉曼散射活性陣列。其功效在於增強底層金奈米粒子的表面增強拉曼散射效應與降低表面銀層的衰退。實驗結果顯示,表面增強拉曼散射強度比沒有銀層修飾金奈米粒子陣列上的表面增強拉曼散射強度,高了約30倍。此策略實際應用於溶液中含有微量水合焦磷酸鈣(CPPD)的檢測上。使用表面增強拉曼光譜檢測含鈣晶體, calcium pyrophosphate dehydrate (CPPD)其拉曼光譜特徵位於741,968,1051,1143,1245,1366 and 1885 cm-1與文獻報告互相吻合。
簡而言之,本研究顯示含鈣晶體會刺激蝕骨前驅細胞分化成熟,並導致骨頭被侵蝕,未來可提供新且有效治療鈣晶體性關節炎的方法,此外表面增強拉曼光譜可檢測含鈣晶體,乃是提供創新方法去診斷含鈣晶體性關節炎。
Calcium-containing arthritis is an inflammatory process which is accompanied by histopathologically characteristic bone destruction. The two most common types of calcium-containing arthritis are calcium pyrophosphate dihydrate deposition disease (CPPD), and basic calcium phosphate (BCP) deposition disease. Bone-resorbing osteoclasts can be matured in vitro by culture of monocyte/macrophage precursors in the presence of macrophage colony stimulating factor (M-CSF) and RANKL. These cells also express proteins that typify the osteoclast lineage, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K, and the vitronectin receptor. However, the mechanisms of bone erosion in chronic calcium-containing arthritis remains unknown.
In clinical examination, patients with calcium-containnig crystal arthritis are diagnosed based on patient''s synovial fluids, which is observed by using polarized light microscopy. However, the sensitivity and accuracy based on this observation are not high enough for exactly diagnosing. Currently, surfaced enhanced Raman spectroscopy (SERS) is best utilised as a tool to distinguish calcium-containing crystals that can be found in synovial fluids.
In this study we would like to explore two part. Part I: to investigate the effects of calcium-containing crystals-hydroxyaptite (HA), β-tricalcium phosphate (ß-TCP), and calcium pyrophosphate dehydrate (CPPD) on osteoclastogenesis and resorption. The aim of the present study was to understand the cellular mechanisms of bone erosion in these diseases focusing on the osteoclast as a potential mediator of this pathologic process. Part II: To investigate surface-enhanced Raman spectroscopy (SERS) to detect CPPD crystals. The aim of the present study is provide a new tool to detect CPPD.
Part I results revealed: Three kinds of calcium-containing crystals significantly enhanced RANKL/M-CSF-induced osteoclast differentiation in RAW264.7 cells evidenced by increase of the number of TRAP-positive MNCs, TRAP activity, and resorption pit formation in a dose-dependent manner. HA, ß-TCP and CPPD treatments significantly enhanced RANKL/M-CSF-induced the mRNA expression of TRAP, cathepsin-K and CTR. Moreover, three kinds of calcium-containing crystals promoted the osteoclast differentiation through activation of MAPKs pathway.
Par II results revealed: Ag possesses the highest molar extinction coefficient in metals, resulting in a stronger surface-enhanced Raman scattering effect than that of Au. However, compared to Au, the serious decay of surface-enhanced Raman scattering enhancement in ambient laboratory air limits its reliable application. In this work, we develop a new strategy for preparing highly surface-enhanced Raman scattering -active Ag films deposited on Au nanoparticle (NP)-decorated SiO2 mask arrays based on electrochemical methods. This method enhanced the surface-enhanced Raman scattering effect of underneath Au NPs as well as on reducing the decay of surface Ag films. Experimental results indicate that the SERs on this developed array exhibits a higher intensity by ca. 30-fold, as compared with the Au NP-based array without the modification of Ag films. This strategy is also practically applicable to the trace detection of solutions containing calcium pyrophosphate dihydrate (CPPD). The characteristic SERS bands of CPPD: 741, 968, 1051, 1143, 1245, 1366 and 1885 cm-1 are basically consistent with those of CPPD crystals reported in the literature.
In summary, calcium-containing crystals could promote osteoclast differentiation and bone resorption through MAPKs. Together with synovial activation, this mechanism may be important in the pathogenesis of destructive arthropathies triggered by calcium-containing crystals. These findings allow the development of new effective therapeutic strategies for calcium-containing arthritis. In addition, SERS can detect CPPD crystals. This strategy is also practically applicable to the trace detection of synovial fluid containing calcium pyrophosphate dihydrate (CPPD), which is a common form of calcium crystals found in articular cartilage.
致謝 (Extend thanks to).......................i
縮寫表 (Abbreviations)........................ii
目錄 (Contents)...............................iv
中文摘要 (Abstract in Chinese)................v
英文摘要(Abstract in English).................viii
緒論 (Introduction)...........................1
研究材料和方法 (Materials and methods)........8
結果 (Results)................................17
討論 (Discussion).............................23
結論和展望(Conclusion and Perspective)........36
參考文獻 (References).........................38
圖表(Tables and Figures ).....................50
附錄 (Appendix)...............................66
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