Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20152
標題: 退化性膝關節炎滑囊液之次級蛋白質體分析
Sub-proteome analysis of synovial fluid from knee joint of degenerative osteoarthritis
作者: 林順智
Lin, Shun-Chih
關鍵字: proteome;蛋白質體學;synovial fluid;degenrative arthritis;knee;滑囊液;退化性膝關節炎;膝關節
出版社: 醫學科技研究所
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摘要: 
退化性膝關節炎普遍發生在60歲以上的老年人,主要是由於關節軟骨損壞所造成,目前已知軟骨細胞對於關節軟骨的損壞扮演重要角色。最近的研究顯示關節損壞的過程中會伴隨著細胞型態的變化,藉由反轉錄-聚合酵素連鎖反應、西方墨點轉漬法、免疫細胞學與生化學等方法,可幫助我們了解關節損壞過程中的基因表現。 但是,目前對於退化性膝關節炎以及其關節軟骨損壞的分子機制,仍有許多未明之處。
蛋白質體學技術,結合高解析力的蛋白質分離技術如二維膠體電泳、質譜儀分析技術以及高速資料庫比對,可以進行大規模的蛋白質鑑定和分析。因此,對於成因複雜的醫學與疾病研究是一項相當有力的分析工具。透過大範圍的蛋白質鑑定與比對,可以完整且快速地分析出對於生物學及臨床醫學有潛在意義的生物標記。此外,文獻顯示透過體液的蛋白質體分析,如血清、尿液、唾液與組織透析液等,可以分離與鑑定出重要的生物標記並進一步分析其生理病理機制。
滑囊液是一種存在滑囊關節中、薄而粘稠的液體。隨著關節軟骨的損壞進程,滑囊液的蛋白質組成會因而改變。近年來風濕性關節炎之軟骨細胞或關節軟骨蛋白質體之研究已有文獻發表;然而,正常與退化性膝關節炎間廣泛的比較性滑囊液蛋白質體研究依舊不多。因此,本實驗之目的在於研究正常與退化性膝關節炎間滑囊液蛋白質體的差異,進而企圖尋找出具潛力的生物標記以及進一步瞭解關節退化進程中的分子機制。藉由二維膠體電泳技術,先以基質輔助雷射脫附-飛行式質譜儀分析技術,快速與大量地進行正常與退化性膝關節炎的滑囊液蛋白質體分析與鑑定。接者將初步鑑定所得之結果,再進一步以液相層析-電灑法質譜儀分析與專一性西方墨點轉漬檢測,進行確認與定量分析。
實驗結果顯示,不同病人之退化性膝關節炎滑囊液蛋白質譜在一維膠體電泳中是非常相似的。此外,初步的二維膠體電泳結果顯示分離效果不佳,推測可能是因為滑囊液中含有大量的肽多糖、透明質酸以及白蛋白與免疫球蛋白等。因此,我們進行多種樣品分析最佳化的分析實驗。結果顯示,透過白蛋白移除程序可以大幅提高二維膠體電泳之分離效果,且再現性很好適合進行比較與半定量分析。滑囊液樣品經過白蛋白移除程序後,在二維電泳圖譜上經分析發現有10個蛋白質點表現量具有顯著差異。我們進一步選擇其中最顯著的兩種蛋白質:甲型抗胰蛋白酶AAT以及脂蛋白元A-I,利用西方墨點轉漬法進行確認與半定量分析。其中甲型抗胰蛋白酶AAT在退化性膝關節炎病人中的滑囊液含量是低於對照組,而脂蛋白元A-I則是高於對照組,這兩組分析結果都具有統計意義。
歸納上述結果,本研究成功地最佳化滑囊液蛋白質的二維膠體電泳分析,並且鑑定出具有顯著差異表現的蛋白質。透過西方墨點轉漬法的確認與半定量分析,顯示甲型抗胰蛋白酶AAT以及脂蛋白元A-I在退化性膝關節炎病人中的滑囊液含量變化,確實具有顯著之統計意義。未來,將可更進一步地探討其他具有變異性之蛋白質,並結合臨床統計來確認應用於臨床分析之潛力,以及進一步瞭解退化性膝關節炎之生理病理分子機制。

Osteoarthritis (OA) is a common disabling human condition in the world, which ultimately results in degeneration of the articular cartilage. It is generally accepted that, at the molecular level, cartilage degeneration is characterized by a general failure of chondrocyte to maintain an appropriate balance between synthesis and degeneration of extracellular matrices (ECM). Besides, it is believed that degeneration of cartilage may result in different protein profiles in synovial fluid (SF). However, the protein profile variation in cartilage or SF accompanying with degeneration is not well studied.
SF is a stringy fluid and contains abundant glycosaminoglycans (GAGs) and albumin in the cavities of synovial joints. The rich GAGs in SF interfere with many protein analyses, e.g. liquid chromatography and isoelectric focusing. For this reason, we use a sub-proteomic approach to investigate the SF protein profiles among healthy and degenerative OA subjects.
In this study, we collected forty-two SF samples from OA patients underwent total knee replacement surgery and four SF samples from non-OA without inflammation patients for surveillance of OA-specific proteins. No obvious difference is presented on SDS-PAGE and two-dimensional (2-D) gel; however, dozens of OA-specific proteins are shown on albumin-depletion 2-D gels. The protein spots with different expression are picked, in situ digested and MALDI-TOF MS-analyzed. Ten potential biomarkers are identified and characterized. Two proteins (Apolipoprotein A-I and A-IV) related with the lipid metabolism was up-regulated in OA group and inversely alpha-1 antitrypsin precursor (AAT), inhibitor of serine protease, was down-regulated in OA group. Semi-quantification of 2-D PAGE discovered the differential expression between OA and non-OA patients. Another seven proteins were first identified and the relation with the development of OA has not been mentioned. They may play an important role of the development of OA pathogenesis but they underlining mechanism need more complete research. Western blot analysis for ATT and Apolipoprotein A-I had confirmed the differential expression as subproteome analysis of 2-D PAGE. It is encouraging to apply the antibody of AAT and Apo A-I clinically for OA diagnosis, early detection and probably the monitoring the response of therapeutic intervention.
URI: http://hdl.handle.net/11455/20152
其他識別: U0005-1508200819102500
Appears in Collections:生物醫學研究所

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