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研究生： 蘇青珮
Ching-pei Su
論文名稱： 由血漿蛋白質吸附與血小板貼附行為探討多孔洞商業高分子薄膜之生物適應性質
Biocompatibility studies of commercial porous polymeric membranes by plasma protein adsorption and platelet adhesion
指導教授： 陳文逸
Wen-yih Chen
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程與材料工程學系
Department of Chemical & Materials Engineering
畢業學年度： 96
語文別： 中文
論文頁數： 116
中文關鍵詞： 血液適應性 、生物適應性 、高分子膜 、蛋白質吸附 、血小板
外文關鍵詞： porous polymeric membranes, biocompatibility, plasma protein adsorption, platelet adhesion
相關次數： 點閱：12  下載：0 
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    • 本研究主要是利用在多孔性高分子膜表面，血漿蛋白質吸附與血小板貼附，與此兩者之間的相互關係，配合膜的親疏水性進行討論，以期能說明多孔膜的生體適應性，以及血漿中蛋白質吸附至多孔膜表面時，可能對血小板貼附所造成的影響。第一個部份為靜態地吸附蛋白質與血小板，並利用單一蛋白質在多孔膜上吸附後，對血小板貼附的影響；第二個部份則是進行多孔膜接觸血漿，在不同時間長短下的蛋白質吸附檢測，並在不同時間吸附蛋白質後進行血小板的貼附實驗。
    在靜態蛋白質吸附實驗中，利用自配製之三種血漿主要蛋白質混合液(fibrinogen、human serum albumun與?-globulin)與離心全血後所得血小板貧乏血漿(PPP)進行蛋白質吸附，因蛋白質的競爭性吸附，與血漿中其他分子對蛋白質的取代作用(Vroman effect)，故在兩種溶液中的蛋白質吸附量，除了容易改變構型與表面作用力較強的fibrinogen之外，並無明顯相關性。血小板吸附實驗則是以離心全血後得富含血小板溶液(PRP)，貼附後以掃瞄式電子顯微鏡(SEM)，觀察血小板的表面形貌與計數貼附量，而比較其與蛋白質吸附的結果，血小板的貼附量與fibrinogen吸附量呈一線性關係；另外在經過單一蛋白質吸附後，進行移除血漿蛋白質後所得之無血漿血小板溶液(serum-free platelet, SFP)的貼附，也可發現 fibrinogen對血小板貼附有正面的影響，因為血小板表面具有糖蛋白(GPIIb/IIIa)，是為與fibrinogen結合的受器。
    而隨時間進行的實驗，利用PPP進行吸附便可清楚觀察到，在平坦的玻璃蓋玻片表面上，蛋白質的吸附符合競爭性吸附的描述，但在表面呈纖維狀，較不平坦的多孔膜則無此現象。而在PPP隨時間吸附後進行SFP中血小板的貼附，觀察到在多孔膜上仍然會有血小板貼附達最大值後，隨時間減少的情形，與蛋白質吸附量量測的結果不同，推測原因為蛋白質在吸附於多孔膜表面後，發生了構型重排，雖保有被特定抗體專一辨識的性質，但已失去與血小板產生連結的功能。

    Surface properties of poymeric biomaterials, such as specific functional groups, charges, and hydrophobicity, play important roles in modulating protein adsorption and cell adhesion on polymeric membranes. Besides, the competitive nature of protein adsorption makes the adsorption behavior and the induced cell adhesion complex. The competitive adsorption of proteins depends upon its molecular weight, bulk concentration of proteins and surface affinity to the proteins.
    The major focus of this study is to investigate the influencing factors for plasma protein adsorption and platelet adhesion on the membrane filters having various kinds of chemical structure and pore size. We found that the adsorption amounts of fibrinogen and ?-globulin decreased while increase of that of human serum albumin (HSA) increased with the membrane hydrophobicity increasing. The human serum albumin was more adsorbed on the membranes from platelet-poor-plasma (PPP) than from mixed protein solution (MPS) consisting of human albumin, ?-globulin and fibrinogen. This tendency was extensively found on hydrophobic membranes compared to the hydrophilic membranes. The number of adhering platelets was lower on membranes with a decreased amount of adsorbed fibrinogen. Suppression of platelet adhesion could be elucidated by a reduction of protein adsorption, in particular of fibrinogen, which bound to the platelet membrane glycoprotein, GP IIb–IIIa. Time-dependent adsorption of fibrinogen indicates that Vroman effect, the displacement of fibrinogen, induced on the flat surface such as glass plates, but was not observed on the porous polymeric membranes.

    目錄 中文摘要 I ABSTRACT III 誌謝 IV 目錄 V 圖目錄 VIII 表目錄 XII 第一章 緒論 1 第二章 文獻回顧 4 2.1 血液相容性材料的探討 4 2.1.1 血漿蛋白質吸附 8 2.1.2 血小板吸附與活化 13 2.1.3白血球的貼附與活化及發炎反應 (inflammatory response) 17 2.2 多孔性高分子膜 23 2.2.1多孔性高分子膜應用在血液透析的發展 23 2.2.2常見高分子膜材料回顧 26 第三章 實驗藥品與儀器設備 30 3.1 實驗藥品 30 3.2 儀器設備 32 3.3 實驗方法 33 3.3.1 PBS緩衝液的製備 33 3.3.2 蛋白質吸附實驗 33 3.3.2.1 血漿溶液的製備 33 3.3.2.2 蛋白質吸附實驗(實驗組) 33 3.3.2.3 蛋白質吸附實驗(控制組) 35 3.3.2.4 不同時間血漿蛋白質的吸附 35 3.3.3 血小板貼附實驗 36 3.3.3.1 血小板溶液的製備 36 3.3.3.2 血小板貼附 36 3.3.3.3單一蛋白質吸附後再進行血小板貼附 37 3.3.3.4血漿蛋白質隨時間吸附後再進行血小板貼附 37 第四章 結果與討論 39 4.1 表面分析 39 4.2 蛋白質吸附實驗 41 4.2.1 水接觸角與蛋白質吸附的關係 41 4.2.2混合蛋白質溶液(Mixed protein solution, MPS)與血小板貧乏血漿(Platelet-poor-plasma, PPP)蛋白質吸附量的關係 43 4.3血小板貼附實驗 50 4.3.1水接觸角、蛋白質吸附與富含血小板血漿(PRP)中血小板貼附的關係 50 4.3.2富含血小板血漿(PRP)中血小板貼附之SEM形貌圖 53 4.3.3蛋白質吸附與血小板貼附之關係 61 4.3.3.1 Fibrinigen對血小板貼附的影響 63 4.3.3.2 HSA對血小板貼附的影響 66 4.3.3.3 IgG對血小板貼附的影響 70 4.4蛋白質不同吸附時間之吸附量 74 4.4.1比較PPP與MPS中蛋白質隨時間的吸附 74 4.4.2 PPP中蛋白質隨時間的吸附後進行血小板的貼附 77 4.4.2.1血漿蛋白質隨時間吸附於PC膜後，血小板的貼附情形 77 4.4.2.2血漿蛋白質隨時間吸附於PTFE膜後，血小板的貼附情形 80 4.4.2.3血漿蛋白質隨時間吸附於玻璃蓋玻片後，血小板的貼附情形 83 第五章 結論 89 第六章 參考文獻 92 圖目錄 圖2-1 凝血機制 7 圖2-2 酵素連結免疫吸附分析法之簡易流程圖 12 圖2-3 血小板的對多項生理機能的調控 14 圖2-4 Foreign body reaction 19 圖2-5 白血球、血小板與內皮細胞之間的相對應受器 20 圖2-6 常見的PEG固定化方法 27 圖3-1 酵素連結免疫吸附分析儀 32 圖4-1 各多孔性高分子膜之靜態水接觸角量測值 39 圖4-2 FN(PPP)吸附與接觸角之關係 42 圖4-3 IgG(PPP)吸附與接觸角之關係 42 圖4-4 HSA(PPP)吸附與接觸角之關係 42 圖4-5 對血漿進行稀釋後測試其吸附量的差異 43 圖4-6 FN於MPS與PPP中吸附量的關係(不同膜材) 44 圖4-7 IgG於MPS與PPP中吸附量的關係(不同膜材) 44 圖4-8 HSA於MPS與PPP中吸附量的關係(不同膜材) 44 圖4-9 FN於MPS與PPP中吸附量的關係(不同親疏水性) 45 圖4-10 IgG於MPS與PPP中吸附量的關係(不同親疏水性) 45 圖4-11 HSA於MPS與PPP中吸附量的關係(不同親疏水性) 45 圖4-12 綜合比較三種蛋白質於MPS與PPP中吸附量的關係 46 圖4-13 Fibrinogen結構示意圖 48 圖4-14 血小板計數方法 50 圖4-15 血小板貼附量與接觸角關係圖 51 圖4-16 血小板貼附量與FN吸附量關係圖 51 圖4-17 血小板貼附量與HSA吸附量關係圖 52 圖4-18 SEM拍攝之血小板貼附表面形貌 53 圖4-19 聚碳酸脂膜(polycarbonate, PC)表面PRP血小板的貼附 54 圖4-20 鐵氟龍(PTFE)表面PRP血小板的貼附 55 圖4-21 聚偏二氟乙烯樹脂膜(PVDF)表面PRP血小板的貼附 58 圖4-22 聚碸(PSf)表面PRP血小板的貼附 59 圖4-23 醋酸脂膜(cellulose acetate, CA)表面PRP血小板的貼附 59 圖4-24 聚碳酸脂膜(PC, 0.2?m)在FN吸附後， PRP中的血小板貼附表面形貌 62 圖4-25 聚碳酸脂膜(PC, 0.2?m)在FN吸附後， SFP中的血小板貼附表面形貌 62 圖4-26 聚偏二氟乙烯樹脂膜(PVDF, 0.22?m)在FN吸附後， PRP中的血小板貼附表面形貌 63 圖4-27 聚偏二氟乙烯樹脂膜(PVDF, 0.22?m)在FN吸附後， SFP中的血小板貼附表面形貌 63 圖4-28 鐵氟龍(PTFE, 0.2?m)在FN吸附後， PRP中的血小板貼附表面形貌 64 圖4-29 鐵氟龍(PTFE, 0.2?m)在FN吸附後， SFP中的血小板貼附表面形貌 64 圖4-30 單一蛋白質吸附3小時後之ELISA吸收值 65 圖4-31 聚碳酸脂膜(PC, 0.2?m)在HSA吸附後， PRP中的血小板貼附表面形貌 66 圖4-32 聚碳酸脂膜(PC, 0.2?m)在HSA吸附後， SFP中的血小板貼附表面形貌 66 圖4-33 聚偏二氟乙烯樹脂膜(PVDF, 0.22?m)在HSA吸附後， PRP中的血小板貼附表面形貌 67 圖4-34 聚偏二氟乙烯樹脂膜(PVDF, 0.22?m)在HSA吸附後， SFP中的血小板貼附表面形貌 67 圖4-35 鐵氟龍(PTFE, 0.2?m)在HSA吸附後， PRP中的血小板貼附表面形貌 68 圖4-36 鐵氟龍(PTFE, 0.2?m)在HSA吸附後， SFP中的血小板貼附表面形貌 68 圖4-37 聚碳酸脂膜(PC, 0.2?m)在IgG吸附後， PRP中的血小板貼附表面形貌 70 圖4-38 聚碳酸脂膜(PC, 0.2?m)在IgG吸附後， SFP中的血小板貼附表面形貌 70 圖4-39 聚偏二氟乙烯樹脂膜(PVDF, 0.22?m)在IgG吸附後， PRP中的血小板貼附表面形貌 71 圖4-40 聚偏二氟乙烯樹脂膜(PVDF, 0.22?m)在IgG吸附後， SFP中的血小板貼附表面形貌 71 圖4-41 鐵氟龍(PTFE, 0.2?m)在IgG吸附後， PRP中的血小板貼附表面形貌 72 圖4-42 鐵氟龍(PTFE, 0.2?m)在IgG吸附後， SFP中的血小板貼附表面形貌 72 圖4-43 MPS中的FN隨時間吸附量 74 圖4-44 PPP中的FN隨時間吸附量 75 圖4-45 MPS中的IgG隨時間吸附量 75 圖4-46 PPP中的IgG隨時間吸附量 75 圖4-47 MPS中的HSA隨時間吸附量 76 圖4-48 PPP中的HSA隨時間吸附量 76 圖4-49 PC膜(0.2?m)先以PPP蛋白質吸附特定時間後進行血小板的貼附 77 圖4-50 PTFE膜(0.2?m)先以PPP蛋白質吸附特定時間後進行血小板的貼附 80 圖4-51 玻璃蓋玻片先以PPP蛋白質吸附特定時間後進行血小板的貼附 84 圖4-52 PTFE膜表面FN吸附與血小板貼附關係於各部份實驗中結果之比較 87 表目錄 表2-1 心血管設備可能引起之併發症 5 表2-2 常見之有機高分子生物材料與其醫療用途 6 表2-3 血液組成 9 表2-4 血小板細胞表面感受器 15 表2-5 白血球的種類 18 表2-6 白血球之表面受器 20 表2-7 白血球表面與發炎反應相關的受器 22 表2-8 1965年前非薄膜式的血液透析器設計 24 表2-9 生醫用膜發展 24 表2-10 常見的表面性質分析方法 28 表3-1 使用膜材之成分、廠牌與孔徑大小。 31 表4-1 Fibrinogen、?-globulin與Human serum albumin的分子量 48 表4-2 血小板於多孔性高分子膜表面貼附數量 51

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