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博碩士論文 etd-0622113-093117 詳細資訊
Title page for etd-0622113-093117
論文名稱
Title
羧基醣化之之牛血清蛋白具破膜活性
Bovine serum albumin with glycated carboxyl groups shows a novel membrane-damaging activity
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
73
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2013-07-10
繳交日期
Date of Submission
2013-07-22
關鍵字
Keywords
破膜活性、醣化作用、脂質體、膜融合
glycation, liposome, membrane fusion, membrane-damaging activities
統計
Statistics
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中文摘要
本研究旨在探討醣化作用對BSA的結構與功能之特性的影響,葡萄糖及p-aminophenyl α-D-mannopyranoside 分別用於製備胺基醣化的AGE-BSA及羧基醣化的Man-BSA。質譜儀分析、HPLC、螢光與CD測量結果顯示醣化作用會改變BSA的物化性質,與AGE-BSA相較,Man-BSA 有著較高的親水性。Man-BSA的內源性螢光強度比BSA及AGE-BSA低,且其結構穩定度高於BSA及AGE-BSA。以螢光測量探討醣化BSA對脂質體的破膜活性、膜擾動效應及膜融合特性,相較於 BSA及AGE-BSA,Man-BSA展現顯著的破膜活性;然而這些蛋白質具有相似脂質體的結合親和力。值得注意的是Man-BSA會導致EYPC/EYSM/Chol脂質體進行膜融合,而增加EYPC/EYSM脂質體膜的通透性,FTIR圖譜顯示在與EYPC/EYSM/Chol或EYPC/EYSM結合時,Man-BSA會採行相異的結構。p-aminophenyl α-D-glucopyranoside修飾羧基的Glc-BSA,同樣地呈現與Man-BSA相似的破膜活性。溶血試驗與MTT分析顯示Man-BSA造成膽固醇剔除的細胞膜通透性增加,並導致細胞死亡。Man-BSA與包含As2O3的EYPC/EYSM/Chol脂質體共同處理時,能增進藥物運輸至癌細胞效率,增加As2O3的細胞毒殺作用。總結來說,本研究指出Man-BSA展現新穎的破膜活性,且膽固醇會影響Man-BSA對磷脂雙層膜的破膜型式。
Abstract
The aim of the present study is to investigate the effect of glycation on structur and functional properties of bovine serum albumin (BSA). Glucose and p-aminophenyl α-D-mannopyranoside were employed to modify -amino groups and carboxyl groups of BSA for preparation of AGE-BSA and Man-BSA, respectively. The results of mass analyses, HPLC, fluorescence and CD measurement indicated that glycation markedly changed physicochemical properties of BSA. Compared with AGE-BSA, Man-BSA had higher hydrophilicity. Fluorescence intensity of Man-BSA was lower than those of native BSA and AGE-BSA. The structural stability of Man-BSA was notably higher than that of BSA and AGE-BSA. The membrane-damaging activity, membrane perturbation effect and fusogenicity of glycated BSA on liposomes were further investigated using fluorescence measurement. In contrast to BSA and AGE-BSA, Man-BSA showed a notable membrane-damaging activity on liposomes, while the binding-affinity of these proteins for liposomes was similar. Noticeably, Man-BSA induced fusion of EYPC/ EYSM/Chol vesicles and membrane permeability of EYPC/EYSM vesicles. FTIR spectra showed that Man-BSA adopted distinct structure upon binding with EYPC/ EYSM/Chol and EYPC/EYSM vesicles. Likewise, Glc-BSA in which carboxyl groups were modified with p-aminophenyl α-D-glucopyranoside showed similar membrane- damaging activities noted with Man-BSA. The results of hemolysis and MTT assay indicated that Man-BSA induced cell death and lysis of cholesterol-depleted cells. Co-treatment of Man-BSA and liposome-encapsulated As2O3 further increased the cytotoxicity of As2O3, resulting from improving As2O3 delivery efficiency into cancer cells. Collectively, our data indicate that Man-BSA displays novel membrane-damaging activities, and that cholesterol crucially regulates the mode of Man-BSA on damaging phospholipid bilayers.
目次 Table of Contents
論文審定書 .................................................................................................................. ii
摘要 ............................................................................................................................. iii
Abstract ...................................................................................................................... iv
目次 .............................................................................................................................. v
圖次 ........................................................................................................................... viii
表次 .............................................................................................................................. x
英文縮寫表 ................................................................................................................. xi
第一章、 緒論....................................................................................................... 1
1.1 醣蛋白的生物功能 ....................................................................................... 1
1.2 Advanced glycation end products (AGEs) .................................................. 2
1.3 醣類修飾在藥物運輸上的運用 .................................................................... 2
1.4 Albumin ....................................................................................................... 3
1.5 研究目的....................................................................................................... 4
第二章、 實驗材料與方法 ................................................................................... 5
2.1 實驗材料....................................................................................................... 5
2.2 製備醣化BSA .............................................................................................. 7
2.3 SDS膠體電泳............................................................................................... 7
2.4 MALDI-TOF質譜儀分析 ........................................................................... 8 2.5 逆向高效能液相層析法 (RP-HPLC) .......................................................... 8
2.6 UV-Vis吸收光譜分析 .................................................................................. 8
2.7 Circular dichroism (CD,Jasco J-810 spectropolarimeter)光譜分析 ....... 8
2.8 蛋白質內生性 (Intrinsic) 螢光測量 ........................................................... 9
2.9 脂質體 (liposome)製備 .............................................................................. 10
2.10 Calcein 螢光釋放分析............................................................................... 11
2.11 膜融合 (fusion)分析實驗 .......................................................................... 12
2.12 FPE螢光分析 ............................................................................................ 12
2.13 Polydiacetylene (PDA)呈色反應 ............................................................... 13
2.14 分析脂質體對蛋白質結構的影響 .............................................................. 14
2.15 溶血試驗..................................................................................................... 14
2.16 細胞培養..................................................................................................... 15
2.17 細胞Calcein-AM (acetoxymethyl ester)釋放分析 .................................... 15
2.18 脂質體與細胞融合 (fusion)實驗 ............................................................... 16
2.19 RT-PCR ..................................................................................................... 16
2.20 細胞存活率檢測 (MTT assay) .................................................................. 17 2.21 As2O3 (Arsenic trioxide)細胞毒性測試 ..................................................... 17
第三章、 結果..................................................................................................... 18
3.1 醣化BSA特性之分析................................................................................ 18
3.2 醣化作用對BSA結構的影響 .................................................................... 19
3.3 醣化作用影響BSA結構的穩定性(stability)............................................. 19
3.4 Man-BSA展現破膜活性 ........................................................................... 20
3.5 Man-BSA對於不同組成的脂質體,導致不同程度的膜融合 .................. 21
3.6 比較不同醣化BSA與脂質膜的結合能力 ................................................. 21
3.7 不同組成的脂質膜會影響Man-BSA的結構 ............................................ 21
3.8 膽固醇影響Man-BSA溶血能力 ............................................................... 22
3.9 剔除膽固醇可增加Man-BSA對癌細胞的毒性(cytotoxicity) .................. 22
3.10 Man-BSA可促進含膽固醇的脂質膜融合 ................................................ 23
3.11 膜蛋白參與Man-BSA與細胞膜的交互作用 ............................................ 23
3.12 Mannan 抑制醣化BSA的破膜活性 ........................................................ 24
3.13 Man-BSA可增進脂質體運輸藥物的效率 ................................................ 25
第四章、 討論..................................................................................................... 26
參考文獻 .................................................................................................................... 55
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