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研究生:黃鈞琬
研究生(外文):Chun-Wan Huang
論文名稱:磁性奈米粒子於抗血管新生之應用
論文名稱(外文):Magnetic Nanoparticles for Target Anti-Angiogenesis
指導教授:馬蘊華
指導教授(外文):Y. H. Ma
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
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:2008
畢業學年度:96
論文頁數:120
中文關鍵詞:磁性奈米粒子血管新生兒茶素
外文關鍵詞:magnetic nanoparticleangiogenesisepigallocatechin-3-gallateEGCG
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透過磁場可引導磁性奈米粒子之特性,利用磁性奈米粒子做為攜藥載體可讓藥物標的作用於病灶處。過去文獻指出綠茶萃取物epigallocatechin-3-gallate (EGCG)具抗氧化、抗血管新生功效,但於生物體內要達到此功效的血中濃度,遠比以飲茶方式攝取EGCG濃度還高。此計劃藉磁性奈米粒子作為攜藥載體,測試磁場是否增強EGCG抗血管新生之功效。實驗結果顯示有聚乙烯二醇(polyethylene glycol,PEG)及葡萄聚糖(dextran)修飾的磁性奈米粒子(MNP-PEG)可進入並滯留於植於大鼠及小鼠體內Matrigel®的新生血管中。利用硫氰酸鉀測鐵法發現磁性奈米粒子亦可被培養皿中的人類血管內皮細胞所攝入,其量明顯較無PEG修飾的粒子少,但磁場不能明顯促進MNP-PEG的攝入。本論文進一步測得EGCG吸附在磁性奈米粒子上的最佳條件,並發現吸附於MNP-PEG上的EGCG仍具抗氧化能力,且MNP-PEG-EGCG之抗氧化活性至少可穩定維持10天。而磁場可促進MNP-PEG-EGCG抑制內皮細胞移行、增生及胸主動脈環微管出芽能力,使閾值以下的EGCG濃度產生作用。磁場的作用很可能是由於促進MNP-PEG-EGCG被內皮細胞攝入而促進EGCG的作用。本論文顯示接合於磁性奈米粒子上之EGCG可能改變磁性奈米粒子的特性,而其抗血管新生作用可因與磁性奈米粒子結合受磁場影響而被促進。
Magnetic nanoparticles have been tested for targeting cancer therapeutics under the guidance of magnetic force. It has been demonstrated that epigallocatechin-3-gallate (EGCG), a component of green tea extract, exerts antioxidant and anti-angiogenesis effect at a pharmacological concentration higher than that can be achieved by drinking green tea. We asked whether magnetic field may enhance the effect of MNP-PEG-EGCG composite in cultured human umbilical cord endothelial cells (HUVEC). Retention of MNP with dextran coating and polyethylene glycol (PEG) modification was demonstrated in the neovasculature of Matrigel® in vivo. In cultured HUVEC exposed to MNP-PEG for up to 24 hr, the amount of MNP-PEG taken up by HUVEC was time-dependent, as measured by a potassium thiocyanate method; however, NdFeB magnet did not enhance MNP-PEG uptake by HUVEC. Adsorption of EGCG with MNP-PEG was conducted and optimal conditions were determined. Stability of MNP-PEG-EGCG composite appeared to be constant up to 10 days, as measured by DPPH assay. Free EGCG attenuated HUVEC migration and proliferation in a concentration-dependent manner. Application of magnet significantly enhanced MNP-PEG-EGCG-induced inhibitory effects on HUVEC migration and proliferation of HUVEC and microvessel sprouting from aortic ring. The augment effects of magnet were likely due to an enhanced MNP-PEG-EGCG uptake by HUVEC. Our results demonstrated potential of EGCG for magnetic targeted anti-angiogenesis.
目錄(Contents)
指導教授推薦書..............................................
口試委員會審定書............................................
授權書..................................................iii
致謝....................................................iv
中文摘要..................................................v
英文摘要.................................................vi
目錄....................................................vii
縮寫表...................................................xi
第一章 緒論(Introduction)..................................1
1. 緒論...................................................1
1.1 腫瘤細胞的血管新生(Tumor-Derived Angiogenesis)..........1
1.2 抗血管新生作為抗癌的治療方式.............................4
1.3 標的給藥(Drug Targeting)..............................5
1.4 奈米醫藥(Nanomedcine).................................7
1.5 磁性奈米粒子在癌症治療的應用............................10
1.6 調理作用的產生及修飾奈米微粒的表面.......................11
1.7 奈米粒子對細胞的毒性...................................16
1.8 EGCG抗血管新生及與磁性奈米粒子結合在癌症治療的應用........17
第二章 實驗目的(Specific Aims)............................22
第三章 實驗材料與方法(Materials and Methods)..............23
1:實驗材料(Materials)...................................23
2:動物品系..............................................24
3:實驗方法(Methods).....................................24
3.1 人類臍靜脈內皮細胞的收集...............................24
3.2 細胞培養(Cell Culture)...............................25
3.3 體外生物活性(In Vitro)層面的磁場給予...................26
3.4 穿透式電子顯微鏡分析磁性奈米粒子在細胞內的分佈............27
3.5 硫氰酸鉀測鐵法(Potassium Thiocyanate Method)..........27
3.6 活體基膠質栓子模型(In Vivo Matrigel Plug Assay)........29
3.7 磁性奈米粒子於基膠質內分佈情形
(Magnetic Nanoparticle Biodistribution in Matrigel)...30
3.8 藥物結合(Drug Conjugation Method)....................31
3.9 磁性奈米粒子粒徑分析(MNP Diameter Analysis)............32
3.10 清除自由基的活性測試
(Free Radical Scavenging Activity Assay--DPPH Assay)..33
3.11 細胞遷移分析(Cell Migration Assay)...................34
3.12 細胞增生分析(MTT assay)..............................35
3.13 胸主動脈環微管出芽模型
(Thoracic Aortic Ring Microvessel Sprouting Model)...36
3.14 統計方法............................................38
第四章 實驗結果(Results).................................39
4.1 磁性奈米粒子可進入組織內新生血管中.......................39
4.2 內皮細胞攝入磁性奈米粒子於細胞質中.......................40
4.3 PEG修飾可減少磁性奈米粒子進入內皮細胞................... 41
4.4 決定製成藥物複合物的最適條件............................42
4.5 藥物複合物粒徑分析.....................................45
4.6 藥物複合物的活性測試...................................45
4.7 藥物複合物穩定性......................................48
4.8 藥物複合物可抑制內皮細胞之移行..........................51
4.9 藥物複合物可抑制內皮細胞之增生能力.......................53
4.10 藥物複合物可抑制胸主動脈環微血管生成....................55
4.11 經吸附EGCG可促進內皮細胞攝入磁性奈米粒子................56
第五章 討論(Discussion)..................................58
5.1 磁性奈米粒子的特性.....................................58
5.2 磁性奈米粒子到達血管新生標的部位的機制...................63
5.3 磁性奈米粒子作為攜藥載體...............................65
5.4 藥物複合物抑制血管新生效價.............................68
第六章 結論(Conclusion)..................................73
參考文獻(Reference)......................................74
附錄(Appendix)...........................................86
圖表(Figure).............................................87
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