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研究生： 趙偉鈞
Chao, Wei-Chun
論文名稱： 以毛細管電泳法分析組織中葡萄胺聚醣之單糖含量
Analysis of Glycosaminoglycan Monosaccharides Composition in Cartilage by Capillary Electrophoresis
指導教授： 張冠諒
Chang, Quan-Liang
學位類別： 碩士
Master
系所名稱： 工學院 - 醫學工程研究所
Institute of Biomedical Engineering
論文出版年： 2006
畢業學年度： 94
語文別： 中文
論文頁數： 58
中文關鍵詞： 酸解 、葡萄胺聚醣 、毛細管電泳 、單糖 、定量分析
外文關鍵詞： Acid Hydrolysis, Quantitative analysis, Monosaccharide, Capillary Electrophoresis, Glycosaminoglycan
相關次數： 點閱：131  下載：1 
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    • 　　葡萄胺聚醣為細胞外基質主要成份之一，它由重複的雙糖重複相連結合成長鏈狀醣類，並與蛋白質形成蛋白醣類存在於細胞外基質中。因為近年來組織工程的發展，人們逐漸重視細胞外基質對生物體在許多方面所扮演的角色。本研究旨再建立葡萄胺聚醣的定量方式，將葡萄胺聚醣經酸解作用後釋出單糖體，反推作為定量該糖的方法。酸解能去除部分醣類上的化學修飾，並且可避免萃取與純化等步驟可能造成的流失，得到相對單純且完整的醣類資訊。利用毛細管電泳靈敏、快速、簡便以及精確等特點，作為研究的分析工具。以具高紫外光吸收值的緩衝溶液作為背景襯托較不吸光的單糖體的電泳方式，可取代有作用不完全之虞的衍生方法，得到忠實的醣類定量資訊。我們以此方式探討葡萄胺聚醣標準品以及猪的透明軟骨組織，其不同的酸解條件對醣類釋放的情形，得到能釋放較多單糖體的酸解環境。本研究並以往年被廣為使用化學呈色方式加以確認此法的可信度，結果顯示本方法與化學方法擁有一致性，而本研究方法能更精確定性以及定量出不同單糖體成份。

    　　Glycosaminoglycan, which is composed of repeated disaccharides, is the major component of the extracellular matrix. It exists by connecting with protein as proteoglycan. As the development of tissue engineering nowadays, the influence of extracellular matrix in many aspects of biological functions are respected. In this study, we aim to establish a quantitative method of glycosaminoglycan. By acid hydrolysis, we can obtain simple monosaccharide component, which has no sulfate group on it. It avoids the procedure of extraction and purification, and has less sample loss. Hence relatively intact information could be attained. Because capillary electrophoresis has outstanding natures of sensitivity, quickness, simple operation, and precision. We chose it to be our analytic tool. Using ultraviolet-absorbing buffer contrasts non-ultraviolet-absorption monosaccharides. We can avoid the chemical derivatization, which could causes uncompleted reaction. These methods are applied to analyze the glycosaminoglycan standard and pig hyaline cartilage. Through different condition of acid hydrolysis, we profile the monosaccharides released from the different sample and find the suitable condition. The result was confirmed by well-established chemical assay. In this study, we establish a method to quantify the monosaccharide of glycosaminoglycan which are in the cartilage. And this methods has more qualitative ability than traditional chemical assay.

    目 錄 中文摘要.................................. I 英文摘要.................................. II 致謝...................................... III 目錄...................................... IV 表目錄.................................... VIII 圖目錄.................................... IX 第一章 簡介............................... 1 1.1 葡萄胺聚醣及其生理意義................ 1 1.2 葡萄胺聚醣成份變化與生物力學的關係.... 6 1.3 傳統糖生理的分析方法.................. 7 1.4 毛細管電泳法的原理與優缺點............ 9 1.5 以毛細管電泳法分析葡萄胺聚醣的現況.... 12 1.6 組織中GAG分析之前處理................. 15 1.7 組織中葡萄胺聚醣成份單糖之製備........ 16 1.8 研究動機.............................. 18 第二章 實驗材料與方法..................... 19 2.1 儀器設備.............................. 19 2.1.1 毛細管電泳分析儀.................... 19 2.1.2 毛細管.............................. 19 2.1.3 偵測器.............................. 19 2.1.4 其他設備............................ 19 2.2 藥品.................................. 21 2.2.1 分析樣品............................ 21 2.2.2 緩衝溶液............................ 21 2.2.3 樣品溶液............................ 22 2.2.4 其它藥品............................ 22 2.3 實驗方法.............................. 23 2.3.1 樣品配製方法........................ 23 2.3.2 電泳液以及緩衝溶液配製方法.......... 23 2.3.3 動物組織處理方法.................... 23 2.3.4 動物組織水分含量測試方法............ 24 2.3.5 組織酸解方法........................ 24 2.3.6 動物組織分析液配製方法.............. 24 2.3.7 標準曲線製作........................ 24 2.3.8 毛細管電泳儀操作條件................ 24 2.3.9 m-Hydroxybiphenyl method 檢測糖醛酸. 25 2.3.10 MBTH method 檢測六碳胺糖........... 26 第三章 組織中單糖之分析結果............... 27 3.1 毛細管電泳條件確立.................... 28 3.1.1 樣品溶劑的選擇...................... 28 3.1.2 電解液中CTAB的濃度.................. 28 3.1.3 偵測靈敏度.......................... 29 3.1.4 再現性.............................. 29 3.1.5 標準曲線............................ 29 3.1.6 波峰專一性.......................... 30 3.2 酸解方式的探討........................ 31 3.2.1 酸的種類............................ 32 3.2.2 使用酸濃度.......................... 34 3.2.3 酸解溫度............................ 37 3.2.4 酸解時間............................ 39 3.3 酸解及毛細管電泳方法於組織中的應用.... 40 3.3.1 組織樣品檢測結果.................... 40 3.3.2 雜質測試............................ 41 3.3.3 確認軟骨組織的酸解條件.............. 42 3.3.4 以化學呈色法鑑定動物組織中葡萄糖醛酸含量................................ 44 3.3.5 內標準樣品酸解測試.................. 45 第四章 討論............................... 46 4.1 現有軟骨葡萄胺聚醣成份分析研究之優缺點 46 4.2 毛細管電泳方法的確效.................. 47 4.3 酸解葡萄胺聚醣標準品.................. 47 4.4 酸解於組織樣品上的應用................ 49 4.5 化學分析方法測試組織醣類.............. 50 4.6 軟骨中的葡萄胺聚醣成份之推估.......... 51 第五章 結論與展望......................... 52 未來研究方向.............................. 53 表目錄 表1.1 人體內多醣類種類、分子量、組成及分佈 17 表3.1 由濃度-矯正面積標準曲線所得之各單醣之公式以及相關係數............... 30 表3.2 毛細管電泳方法分析猪透明軟骨單糖成份 43 表3.3 傳統化學呈色方法分析猪透明軟骨單糖成份.................................. 44 圖目錄 圖1.1 生物體生理控制示意圖................ 1 圖1.2 細胞外基質以及葡萄胺聚醣結構........ 3 圖1.3 葡萄胺聚醣雙糖結構式................ 5~6 圖3.1 研究架構............................ 28 圖3.2 不同單糖體的濃度標準曲線............ 30 圖3.3 四單糖標準品混和的液毛細管電泳圖譜.. 31 圖3.4 以0.9N硫酸酸解軟骨組織的毛細管電泳圖譜.................................. 32 圖3.5 以2M三氟乙酸酸解軟骨組織的毛細管電泳圖譜................................ 33 圖3.6 以4N鹽酸酸解軟骨組織的毛細管電泳圖譜 34 圖3.7 2N鹽酸酸解軟骨膠硫酸鹽標準品........ 34 圖3.8 4N鹽酸酸解軟骨膠硫酸鹽標準品........ 35 圖3.9 6N鹽酸酸解軟骨膠硫酸鹽標準品........ 35 圖3.10 使用2N鹽酸酸解透明質酸標準品....... 36 圖3.11 4N鹽酸酸解透明質酸標準品........... 36 圖3.12 6N鹽酸酸解透明質酸標準品........... 37 圖3.13 85℃酸解軟骨膠硫酸鹽標準品單糖釋放情形............................... 37 圖3.14 100℃酸解軟骨膠硫酸鹽標準品單糖釋放情............................... 38 圖3.15 85℃下酸解透明質酸標準品單糖釋放情形................................. 38 圖3.16 100℃酸解透明質酸標準品單糖釋放情形 39 圖3.17 以4N鹽酸於85℃溫度下酸解軟骨膠硫酸鹽其單糖釋放百分比............. 40 圖3.18 以4N鹽酸於85℃溫度下酸解透明質酸其單糖釋放百比......................... 40 圖3.19 軟骨組織酸解後之毛細管電泳圖譜..... 41 圖3.20 第二型膠原蛋白經鹽酸處理後之毛細管電泳圖譜............................. 42 圖3.21 第二型膠原蛋白經鹽酸處理後之毛細管電泳圖譜............................. 42 圖3.22(a) 不同濃度的鹽酸作用酸解軟骨組織情形................................. 43 圖3.22(b) 不同濃度的鹽酸作用酸解軟骨組織情形................................. 43 圖3.23 葡萄糖醛酸標準品化學分析標準曲線... 44 圖3.24 葡萄胺糖酸標準品化學分析標準曲線... 44

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