臺灣博碩士論文加值系統

聚羥基烷酯類( Polyhydroxyalkanoates, PHAs )為生物可分解性材料之一，物化特性與傳統塑膠相近，具有取代傳統塑膠的潛力，PHA在本實驗室先前的研究上指出小鼠皮下植入PHA薄膜在6-8個月之後具有明顯的降解情形，而體外評估PHA與細胞株互動和活體植入的結果均顯示，PHA具有不錯的組織相容性可發展為生醫材料。儘管如此，生醫材料若要運用於循環系統上時，還須具備血液相容性的特質。血液相容性差的生醫材料是指材料與血液接觸時會引起血中細胞與組織物質特性的改變，例如：血清凝血系統蛋白質的活化、血小板的凝集和血栓的形成，因此不易造成血栓的生醫材料即是血液相容性較高的材料。現今有關PHAs血液相容性的文獻報導仍不多，因此，本研究的目的是利用血球細胞的貼附測定、紅血球溶解分析測定、血小板再鈣化的時間的測定、血小板吸附與凝集反應的測定等方式配合免疫螢光染色和電子顯微鏡的觀察，對PHB與PHBV高分子做血液相容程度的評估。結果顯示當PHAs高分子接觸血漿中蛋白、血小板或血球時會引發血栓生成的一連串反應，因此其血液相容性並不高，其中PHB的血液相容性比PHBV稍佳，研究中也利用將PHB注入尾部靜脈阻斷的小鼠活體實驗來評估PHB高分子於活體血液相容性的結果。由活體實驗顯示，未改質的PHB懸浮液靜脈注射可引發小鼠尾部尖端血液的栓塞，進而引起小鼠尾部壞死的現象。因此針對PHB進行接枝的改質處理，改質後的PHB薄膜利用相同技術作評估，並運用改質PHB與巨噬細胞或T淋巴球體外共同培養方式，評估改質前後PHB對血液細胞活性的影響，結果顯示表面經過肝素功能基的接枝改質可提昇PHB高分子的血液相容性。綜合以上結果，PHAs血液相容性較低，但表面經過改質後將可提昇其血液相容的特性。

Polyhydroxyalkanoates（PHAs）are bio-degradable materials that possess the similar physical and chemical properties as petrochemical plastics. PHAs have potential to replace the petrochemical plastics. It has been reported that the PHAs implants in animals were starting to degrade on 6-8 months after implantation. It also reveals that PHAs possess high biocompatibility and suit for biomedical materials. Blood-compatibility (also named as hemocompatibility) is major criteria for biomaterials to use in the circulation system of body. The blood-compatibility is defined that the material is nonthrombongenesis and less activities on leukocyte adhesion and activation when materials are interacted with blood, blood vessel, or heart. When the biomaterial is possessed the higher blood-compatibility, it is showed the less serologic and thrombogenic responses after contact with blood components. However, the study on PHA hemocompatibility is quite few, hence, the purpose of this study is evaluated the blood-compatibility on PHA polymers and further developed the methodologies on PHAs’ hemocompatibility mordification. In this study, the hemolytic analysis, platelet and leukocyte adhesion assay, platelet aggregation assay, plasma recalcification time measurement were used to estimate PHAs’ hemocompatibility. The results of in vitro cultured PHA polymers with leukocytes, platelet, and erythrocytes of blood indicated that PHAs possessed poor blood-compatibility. The PHBV were worse than PHB. Furthermore, the administration of unmodified PHB into mice tail vein induced the outcomes of thrombogenesis and necrosis on the end of mouse tail. However, the blood-compatibility of PHBs were improved by heparin graft on PHB film surface used both Poly-(acrylic acid) and Poly-(glycidyl methacrylate) as linkers. In conclusion, this study suggested that PHA polymers possess poor blood-compatibility, but the hemocompatibility of PHAs can be improved after heparin-graft surface modification.

中文摘要 Ⅷ
英文摘要 Ⅸ
第一章、研究背景 1
第一節、生物可分解性高分子 2
1.1.1、定義 2
1.1.2、分類 2
1.1.3、應用 3
第二節、聚羥基烷酯類PHA 3
1.2.1、PHA的結構 3
1.2.2、物理特性 4
1.2.3、化學特性 4
（1）、生物可分解性（biodegradable） 4
（2）、生物可吸收性（Bioresorbable） 5
第三節、PHA的生物相容性（Biocompatibility） 6
1.3.1、組織相容性 6
（1）、組織相容性的定義 6
（2）、文獻報導 6
1.3.2、血液相容性 9
（1）、血液相容性的定義 9
（2）、血液相容性的評估方法 9
（3）、PHAs血液相容性的研究報導 11
第四節、PHAs的應用 11
第五節、具血液相容性之生醫材料及其表面改質 12
第二章、研究動機和目的 14
第三章、實驗設計材料與方法 15
第一節、實驗設計 15
第二節、實驗材料 17
3.2.1、PHAs高分子材料 17
3.2.2、細胞株 17
3.2.3、實驗小鼠 18
第三節、藥品 18
第四節、細胞培養液與各類試劑 20
3.4.1、RPMI-1640 細胞培養液 20
3.4.2、HG-DMEM 細胞培養液 21
3.4.3、細胞增殖反應試藥 21
3.4.4、一氧化氮試藥 21
3.4.5、細胞激素試藥 22
3.4.6、細胞流質儀試劑 23
3.4.7、Tyrode buffer 23
3.4.8、ACD溶液 24
3.4.9、MDA assay buffer preparation 24
第五節、儀器設備 25
第六節、實驗方法與步驟 26
3.6.1、PHAs材料的製備 26
（1）、PHB、PHBV-5、PHBV- 8、PHBV-12薄膜的製備 26
（2）、Polystyrene（PS）薄膜製備 27
3.6.2、PHAs高分子薄膜的改質 27
（1）、薄膜電漿接枝聚合 27
（2）、共價鍵結固定功能基 28
3.6.3、薄膜的材切與滅菌 29
3.6.4、血液相容性的評估 29
（1）、血小板吸附程度的評估 29
A、免疫螢光染色測定 29
B、掃描式電子顯微鏡測定 30
（2）、血小板吸附程度的評估 31
A、血小板凝集反應的測定 31
B、MDA assay 31
（3）、血小板再鈣化的時間測定 32
（4）、白血球貼附程度的評估 32
（5）、紅血球溶解分析測定 33
3.6.5、高分子材料對血液型細胞活性的評估 33
（1）、PHAs高分子對巨噬細胞活性影響的評估 33
A、RAW264.7細胞株的培養 33
B、PHAs與RAW264.7的作用 34
（2）、PHAs高分子對周邊血中淋巴球增殖作用影響 34
A、人類周邊血單核性白血球的分離與培養 34
B、PHAs與PBMC的作用 35
（3）、細胞型態與活性分析 35
A、細胞型態觀察 35
B、細胞增殖活性的測定 35
C、細胞釋放NO功能性測定 36
D、細胞激素釋放的測定 36
3.6.6、PHAs血液相容性之小鼠活體評估 37
3.6.7、統計分析 38
第四章、結果 39
第一節、PHAs高分子材料血液相容性評估實驗 39
（1）、血小板活性測定 37
（2）、白血球細胞的貼附 40
（3）、紅血球溶解分析測定 40
（4）、血漿再鈣化的時間測定 41
第二節、PHB高分子材料改質後血液相容性評估實驗 42
4.2.1、聚丙烯酸為架橋接枝改質 42
（1）、血小板活性測定 42
（2）、白血球細胞的貼附 43
（3）、紅血球溶解分析測定 43
（4）、血漿再鈣化的時間測定 44
4.2.2、聚甲基丙烯酸環氧丙酯為架橋接枝改質 44
（1）、血小板活性測定 44
（2）、白血球細胞的貼附 45
（3）、紅血球溶解分析測定 45
（4）、血漿再鈣化的時間測定 46
4.2.3、Heparin和Chitosan改質後PHB對體外細胞株與人類周
邊血單核性細胞功能性評估實驗 46
（1）、小鼠巨噬細胞株Nitrite與細胞激素釋放的影響 46
（2）、人類周邊血單核性細胞增殖與細胞激素釋放的影響 48
第三節、PHB高分子材料尾部靜脈注射血液相容性評估實驗 49
第五章、討論 51
參考文獻 57
附表目錄
頁
表一、血液相容性評估表 65
表二、ISO 10993 66
表三、ISO 10993-4 67
表四、改質PHB高分子對AA所引發兔子血小板凝集反應影響的分析 68


附圖目錄
頁
圖ㄧ、PHAs膜植入小鼠肝臟的凝血反應 9
圖二、鐘罩式電漿反應器示意圖 28
圖三、血小板凝集反應 31
圖四、人類血小板對PHAs材料吸附與分布狀況的免疫螢光染色圖 69
圖五、人類血小板在PHAs材料上貼附與分布狀況的掃描式電顯圖 70
圖六、PHB高分子對AA所引發兔子血小板凝集反應影響的分析 71
圖七、PHB高分子對AA與兔子血小板作用產生MDA反應影響的分析 72
圖八、人類血中白血球細胞在PHAs材料上貼附狀態的評估 73
圖九、PHAs高分子對紅血球溶解作用影響的評估 74
圖十、PHAs高分子對血漿再鈣化活性影響的評估 75
圖十ㄧ、PHB高分子對小鼠尾部靜脈注射之血液相容性活體評估 76
圖十二、人類血小板在聚丙烯酸/肝素或聚丙烯酸/幾丁聚醣改質之PHB
材料上貼附狀況的電子顯微鏡圖 77
圖十三、人類白血球細胞在經聚丙烯酸/肝素或聚丙烯酸/幾丁聚醣之
PHB材料上貼附狀況的評估 78
圖十四、聚丙烯酸/肝素或聚丙烯酸/幾丁聚醣改質PHB高分子對紅血球的
溶血活性影響的分析 79
圖十五、聚丙烯酸/肝素與聚丙烯酸/幾丁聚醣改質後PHB高分子對血漿
再鈣化活性影響的評估 80
圖十六、血小板豐富血漿在聚甲基丙烯酸環氧丙酯/肝素或幾丁聚醣改質
之PHB材料上貼附狀況的掃描電子顯微鏡圖 81
圖十七、人類白血球細胞在經聚甲基丙烯酸環氧丙酯/肝素或幾丁聚醣之
PHB材料上貼附狀況的評估 82
圖十八、聚甲基丙烯酸環氧丙酯/肝素或幾丁聚醣改質PHB高分子對
紅血球的溶血活性影響的分析 83
圖十九、聚甲基丙烯酸環氧丙酯/肝素或幾丁聚醣改質後PHB高分子
對血漿再鈣化活性影響的評估 84
圖二十、評估改質後PHB高分子對小鼠巨噬細胞RAW264.7釋放NO
能力的影響 85
圖二十ㄧ、評估改質後PHB高分子對小鼠巨噬細胞RAW264.7釋放IL-6
的影響 86
圖二十二、人類周邊血單核細胞在接枝改質後PHB高分子材料上細胞
增殖能力指數（Stimulating Index）的評估 87
圖二十三、改質後PHB高分子對人類周邊血單核細胞釋放IFN-γ能力
的評估 88

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