臺灣博碩士論文加值系統

本論文藉由蛋白質體學的方法，利用streptozotocin (STZ)所引發的糖尿病模式鼠，研究糖尿病相關的血漿蛋白質。高脂肪飲食搭配低劑量STZ誘發的糖尿病大鼠及正常大鼠將測量其血糖值和血糖調控狀況，並且進行總蛋白質體以及親合性層析法所得之磷酸化蛋白質體、醣蛋白質體的蛋白質體分析。在糖尿病鼠的血液中，白蛋白(albumin)、維生素D結合前趨蛋白(vitamin D-binding protein precursor)和甲狀腺素運送蛋白(transthyretin) 表現量減少，磷酸化的phosphorylated- 補體抑制因子H(complement inhibitory factor H)、血漿白蛋白(serum albumin)和alpha-2-HS-glycoprotein表現量增加，醣基化的纖維蛋白原B (fibrinogen B beta chain)、脫酸酯脢前趨蛋白(carboxylesterase precursor)和絲胺酸胜肽抑制劑(serine peptidase inhibitor)表現量增加，本論文將會探討這些被鑑定到在生理上有差異的蛋白質與糖尿病之間的關係。另外，蛋白質體學的技術也利可用於偵測奈米金所引起的細胞死亡機制，本論文發現奈米金可引發慢性骨髓性血癌細胞株(K562)的細胞凋亡，藉由搭配蛋白質體學與生物資學的分析，本論文推測內質網壓力可能為奈米金引發細胞死亡之細胞內的主要事件。

In this study, we utilized proteomic approaches to investigate the differentially expressed plasma protein related to diabetes. The streptozotocin (STZ) diabetic rats were utilized for investigation. Using high fat diet to induce the diabete-like phenotype, the STZ treated and the control rat were examined for the blood sugar level and regrouped for proteomic analysis. By employing our developed affinity chromatography, the total proteome as well as the phosphoproteome and glycoproteome from the rat plasma samples were purified and examined. Briefly, albumin, vitamin D-binding protein precursor, and transthyretin were down- regulated, phosphorylated- complement inhibitory factor H, serum albumin, and alpha-2-HS-glycoprotein were up- regulated, and glycosylated –fibrinogen B beta chain, carboxylesterase precursor, serine peptidase inhibitor were up-regulated in the plasma of diabeteic rats. The physiological significance of the identified proteins with diabetes was discussed. On the other hand, proteomic techniques were also utilized to inspect the mechanism of death elicited by Nanogold particles, which was found effectively inducing apoptosis in human chronic leukemia cells (K562). In conjunction with proteomic techniques and systems biology analysis, we suggest that endoplasmic reticulum (ER) stress response may be the major cellular event elicited by Nanogold particles and induce cell death if unmanageable.

目錄
謝 誌
中文摘要
英文摘要
第一章 概論
1.1 蛋白質體學 (Proteomics)
1.1.1 磷酸化蛋白質體 (Phosphoproteome)
1.1.2 醣蛋白質體 (Glycoproteome)
1.1.3 膜蛋白質體 (Membrane proteome)
1.2 次蛋白質體學的重要性及研究現況
1.3 醣化蛋白質純化
1.3.1 凝集素親和性層析法 (Lectin affinity chormatography)
1.3.2 共價鍵作用力層析法 (Covalent interaction chromatography)
1.4 膜蛋白質純化
1.4.1 超高速離心法 (Ultracentrifugation)
1.4.2 化學萃取法 (Chemical based extraction)
1.5 糖尿病
1.6 慢性骨髓性血癌
1.7 實驗動機與目的
第二章 材料與方法
2.1 細胞破裂法 (Freeze and thaw)
2.2 膜蛋白萃取法 (Membrane purification)
2.3 蛋白質沉澱 (Protein precipitation)
2.4 蛋白質定量 (Protein quantization)
2.4.1 Bradford 定量法
2.4.2 BCA 定量法
2.5 蛋白質純化 (Proteim purification)
2.5.1 固定金屬離子親和層析法(IMAC)
2.5.2 Lectin affinity chromatography
2.5.3 共價鍵作用力層析法 (Covalent interaction chromatography)
2.6 電泳檢定法 (Electrophoresis)
2.7 CyDye 蛋白質螢光標定
2.8 二次元膠體電泳 (Two-dimensional electrophoresis, 2-DE )
2.8.1. 第一次元電泳 (Isoelectric focusing, IEF)
2.8.2 IPG 膠體平衡反應 (IPG strip equilibration)
2.8.3 第二次元電泳 (SDSPAGE)
2.9 膠體染色
2.9.1 鋅染色 (Zinc stain)
2.9.2 SYPRO Ruby 染色
2.9.3 Pro-Q Diamond 染色
2.9.4 Pro-Emerald 300 染色
2.9.1 醣蛋白銀染色 (過碘酸-硝酸銀法)
2.10 影像擷取與分析
2.11 蛋白脢水解
2.12 蛋白質免疫轉印法
2.13 酵素免疫染色
2.14 細胞培養
2.15 細胞存活率之計算
2.16 PI/Annexin V 雙染色法
2.17 蛋白質晶片分析
第三章 結果與討論
3.1 評估醣蛋白層析法之效益
3.1.1 醣染色法及測試樣品之選擇
3.1.2 純化條件測試
3.1.3 不同之醣蛋白親和性層析法比較
3.1.4 反應時間測試
3.2 評估建立細胞膜蛋白質體之條件
3.2.1. 各種介面活性劑對於純化細胞膜蛋白之效益
3.2.2. 評估膜蛋白質體純化效益
3.3 應用於建立糖尿病模式鼠之蛋白質體及次蛋白質體
3.3.1 血液總蛋白質體分析
3.3.2 血液磷酸化蛋白質體
3.3.3 血液醣蛋白質體
3.3.4 肝臟之各蛋白質體
3.4 奈米金對慢性骨髓性血癌細胞之作用機制
3.4.1 奈米金及抗葉酸藥物對慢性骨髓性血癌細胞的影響
3.4.2 細胞死亡之方式
3.4.3 以蛋白質體學方法探討毒殺細胞之機制
3.4.4 IPA (Ingenuity's Pathway Analysis)分析可能之蛋白質訊息傳導路徑
第四章 結論
4.1 醣蛋白之純化
4.2 細胞膜蛋白質體
4.3 糖尿病模式鼠血液之蛋白質體及次蛋白質體
4.4 奈米金對慢性骨髓性血癌細胞之作用機制
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