臺灣博碩士論文加值系統

本研究以天然生物性高分子聚(3-羥基丁酸酯-共-3-羥基己酸酯) (PHBHHx)與纖維連接蛋白嘗試建構生物性膜材。仿造天然細胞生長環境以進行C3A細胞之培養，期能增進細胞之生長與功能。結果顯示，臭氧化PHBHHx膜材，其平均粗糙度從330 ± 22 nm下降至137 ± 3 nm，再接枝丙烯酸的膜材之後並沒有造成太大的變化，其平均粗糙度為143 ± 4 nm。在alamarBlue與MTT檢測結果顯示，C3A細胞培養在這些膜材上展現類似的細胞生長與代謝活性。 C3A細胞生長於丙烯酸接枝與丙烯酸接枝後纖維連接蛋白塗布之PHBHHx膜材上，其細胞分泌albumin的含量有明顯的提升。由SEM觀察C3A細胞生長於丙烯酸接枝、纖維連接蛋白塗布與丙烯酸接枝後纖維連接蛋白塗布之PHBHHx膜材上之形態是以三維的方式聚集生長，也因此可能造成C3A細胞功能之提升。相對於C3A細胞生長於未改質PHBHHx膜材，其形態則是以二維的方式平面生長。

The objective of this study is to mimic the natural cell environment on polymer surface for C3A cell culture. In this study, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and fibronectin were constructed as biofunctional membranes. AFM data and SEM image showed that the surface roughness of the PHBHHx membrane decreased after ozone treatment, but did not change significantly after grafting with acrylic acid (AA). The root-mean-square surface roughness of the unmodified PHBHHx surface was about 330 ± 22 nm. When the PHBHHx surface was treated with ozone for 60 min, the surface roughness decreased to 137 ± 3 nm. After grafting with AA, the surface roughness was 143 ± 4 nm. The alamarBlue test showed that C3A cells exhibited similar metabolic activity on those membranes and the MTT test revealed that cells exhibited good growth activity up to 9th day. The secretion of albumin from C3A cells significantly increased when they cultured on the AA grafted and fibronectin immobilized PHBHHx membranes. The morphology of C3A cells was spheroid-like on the AA grafted and fibronectin immobilized membranes and the three- dimensional (3D) morphology might promote the functions of C3A cells. In contrast, the morphology of C3A cells on unmodified membranes was two-dimensional (2D) monolayer.

目錄
摘要 I
Abstract II
第一章 緒論 1
1.1研究動機與介紹 1
1.2組織工程 2
1.2.1簡介 2
1.2.2細胞與材料分界面的機制 5
1.3研究範疇 8
第二章 研究方法與原理 10
2.1 聚羥基烷酯(polyhydroxyalkanoates, PHAs) 10
2.1.1簡介 10
2.1.2 PHA材料於組織工程之應用 12
2.2纖維連接蛋白(fibronectin)簡介 14
2.3臭氧(Ozone) 15
2.3.1簡介 15
2.3.2臭氧的產生 17
2.4肝細胞(Hepatocyte) 19
2.5細胞骨架(cytoskeleton) 21
2.5.1細胞骨架自行組裝與動態結構 21
2.5.2建構細胞骨架 23
2.6膜材表面過氧化物(peroxide)定量之分析 24
2.6.1碘化物測量法(Iodide method) 24
2.7膜材表面羧基(carboxylic group)定量之分析 25
2.7.1 Toluidine Blue O (TBO) 25
2.8膜材親疏水性之測試 27
第三章 實驗方式 28
3.1 實驗藥品 28
3.2 實驗儀器 30
3.3 試劑配製 32
3.3.1 碘化鈉(Sodium iodide, NaI)溶液的配製 32
3.3.2 Toluidine Blue O (TBO)染劑的配製 32
3.3.3 TBO檢量線溶液的配製 32
3.3.4丙烯酸(acrylic acid)單體溶液的配製 32
3.3.5 培養液配製 33
3.3.6 磷酸緩衝溶液(phosphate buffer solution, PBS) 33
3.3.7 戊二醛溶液配製 34
3.3.8 鋨酸溶液 34
3.4 PHBHHx膜材製備 34
3.5 臭氧處理 (ozone treatment) 35
3.5.1 簡介 35
3.5.2 實驗步驟 35
3.6 過氧化物(peroxide)定量之分析 36
3.7 丙烯酸接枝反應(grafting reaction) 38
3.8 羧基(carboxylic group)定量之分析 38
3.9 親疏水性之測試 40
3.10 膜材前處理 40
3.10.1 簡介 40
3.10.2消毒程序 40
3.11 C3A細胞株的培養 41
3.12細胞繼代 (passage) 41
3.12.1簡介 41
3.12.2細胞繼代步驟 42
3.13 細胞計數與染色 43
3.13.1 簡介 43
3.13.2細胞計數步驟 43
3.14 alamarBlue分析C3A細胞代謝能力(metabolic activity) 44
3.14.1 簡介 44
3.14.2 實驗步驟 44
3.15 MTT分析C3A細胞生長活性(growth activity) 45
3.15.1簡介 45
3.15.2實驗步驟 45
3.16 C3A細胞代謝物測試 47
3.16.1 簡介 47
3.16.2 Ammonia含量分析 47
3.16.3 Albumin含量分析 49
3.17細胞形態之觀察 51
3.17.1相位差倒立式光學顯微鏡 51
3.17.2掃描式電子顯微鏡 (SEM) 51
3.17.2.1簡介 51
3.17.2.2細胞固定與脫水 52
3.17.2.3臨界點乾燥 (critical point drying, CDP) 53
3.18原子力顯微鏡 (AFM) 55
3.18.1 簡介 55
3.18.2 操作步驟 55
3.19 膜材表面電子顯微鏡之觀察 55
3.19.1 簡介 55
3.19.2 實驗步驟 56
第四章 結果與討論 57
4.1 PHBHHx膜材 57
4.1.1 溶鑄法製備膜材 57
4.1.2 膜材表面結構觀察與粗糙度分析 57
4.2 臭氧對於PHBHHx膜材表面過氧化物形成之影響 59
4.3 臭氧對於PHBHHx膜材接枝丙烯酸之影響 59
4.4 丙烯酸單體濃度改變之影響 60
4.5 膜材表面性質分析 60
4.6 膜材表面親疏水性質之影響 60
4.7 C3A細胞代謝活性分析 61
4.8 C3A細胞生長活性分析 61
4.9 C3A細胞功能性分析 62
4.9.1 C3A細胞分泌albumin含量之分析 62
4.9.2 C3A細胞產生ammonia含量之分析 63
4.10 C3A細胞生長形態之觀察 64
4.10.1 OM形態之觀察 64
4.10.2 SEM形態之觀察 65
第五章 結論 99
第六章 參考文獻 101
















表目錄
Table 1.1 蛋白質中所含有特定的胺基酸序列 7
Table 2.1 多種PHA的物理性質與日常生活中所使用的塑膠 14
Table 4.1 臭氧化與丙烯酸接枝PHBHHx膜材 67
Table 4.2 PHBHHx膜材經由不同時間臭氧處理 68














圖目錄
Fig. 1.1 環境訊息的改變影響細胞生長形態 4
Fig. 1.2 細胞與高分子 4
Fig. 1.3 細胞利用特定的穿膜接收器integrin 7
Fig. 2.1.1 The structure of PHAs 11
Fig. 2.1.2 The structure of PHBV. 11
Fig. 2.1.3 The structure of PHBHHx. 11
Fig. 2.1.4 其他由PHAs所製成以應用於移植材料之結構 13
Fig. 2.1.5 由PHBHHx材料所製成的人工食道 14
Fig.2.3.1 Ozone resonance structure. 16
Fig.2.3.2 Observed ozone structure. 16
Fig. 2.3.3 Reaction of ethylene with ozone 17
Fig. 2.3.4 臭氧形成之反應式 18
Fig. 2.3.5 藉由電暈放電方式(corona discharge method)作為產生臭氧之操作示意圖 19
Fig. 2.4.1 肝臟的構造 20
Fig. 2.5.1 三種細胞骨架的類型 22
Fig. 2.5.2 細胞骨架與形狀的改變 24
Fig. 2.6.1 The reaction of iodide ion with peroxide. 25
Fig. 2.6.2 The reaction of iodine with iodine. 25
Fig. 2.7.1 The structure of TBO 26
Fig. 3.1 I3-濃度與可見光吸收波長360 nm之檢量線關係圖 37
Fig. 3.2 TBO濃度與可見光吸收波長633 nm之檢量線關係圖 39
Fig. 3.3 C3A細胞數與O.D.值(570 nm)之檢量線關係圖 46
Fig. 3.4 Ammonia濃度與O.D.值(630 nm)之檢量線關係圖 48
Fig. 3.5 Albumin濃度與O.D.值(405 nm)之檢量線關係圖 50
Fig. 4.1 溶鑄法所製備的PHBHHx膜材 69
Fig. 4.2 溶鑄法製備的PHBHHx膜材，以SEM觀察其表面結構 70
Fig. 4.3 臭氧化PHBHHx膜材，以SEM觀察其表面結構 71
Fig. 4.4 丙烯酸接枝PHBHHx膜材，以SEM觀察其表面結構 72
Fig. 4.5 溶鑄法製備的PHBHHx膜材，以AFM分析其表面粗糙度 73
Fig. 4.6 臭氧化PHBHHx膜材，以AFM分析其表面粗糙度 74
Fig. 4.7 丙烯酸接枝PHBHHx膜材，以AFM分析其表面粗糙度 75
Fig. 4.8 臭氧對烯烴聚合物降解的影響 75
Fig. 4.9 PHBHHx膜材表面生成過氧化物濃度與臭氧處理時間之關
係圖 76
Fig. 4.10 PHBHHx膜材表面過氧化物生成示意圖 76
Fig. 4.11 PHBHHx膜材表面接枝丙烯酸含量 77
Fig. 4.12 PHBHHx膜材表面過氧化物與丙烯酸產生接枝反應示意圖 77
Fig. 4.13 PHBHHx膜材接枝丙烯酸含量與單體濃度之關係圖 78
Fig. 4.14 C3A細胞培養於不同膜材上，顯示其細胞代謝活性 (metabolic activity) 79
Fig. 4.15 C3A細胞培養於不同膜材上，顯示其細胞生長活性 (growth activity) 80
Fig. 4.16 C3A細胞培養於不同膜材上，顯示其細胞分泌albumin含量之功能性表現 81
Fig. 4.17 C3A細胞培養於不同膜材上，顯示每單位(104個)細胞分泌albumin含量之功能性表現 82
Fig. 4.18 C3A細胞培養於不同膜材上，顯示其細胞產生ammonia含量之功能性表現 83
Fig. 4.19 C3A細胞培養於不同膜材上，顯示每單位(104個)細胞產生ammonia含量之功能性表現 84
Fig. 4.20 C3A細胞分別培養於(a)PHBHHx(b)丙烯酸接枝PHBHHx(c)
纖維連接蛋白塗布之PHBHHx (d) 丙烯酸接枝後纖維連接蛋白塗布之PHBHHx膜材，第1天時間點下其細胞生長形態之觀察 86
Fig. 4.21 C3A細胞分別培養於(a)PHBHHx(b)丙烯酸接枝PHBHHx(c)纖維連接蛋白塗布之PHBHHx (d) 丙烯酸接枝後纖維連接蛋白塗布之PHBHHx膜材，第6天時間點下其細胞生長形態之觀察 88
Fig. 4.22 C3A細胞分別培養於(a)PHBHHx(b)丙烯酸接枝PHBHHx(c)
纖維連接蛋白塗布之PHBHHx (d) 丙烯酸接枝後纖維連接蛋白塗布之PHBHHx膜材，第9天時間點下其細胞生長形態之觀察 90
Fig. 4.23 C3A細胞培養於PHBHHx膜材 91
Fig. 4.24 C3A細胞培養於丙烯酸接枝PHBHHx膜材 92
Fig. 4.25 C3A細胞培養於纖維連接蛋白塗布之PHBHHx膜材 93
Fig. 4.26 C3A細胞培養於丙烯酸接枝後纖維連接蛋白塗布 94
Fig. 4.27 C3A細胞培養於PHBHHx膜材 95
Fig. 4.28 C3A細胞培養於丙烯酸接枝PHBHHx膜材 96
Fig. 4.29 C3A細胞培養於纖維連接蛋白塗布之PHBHHx膜材 97
Fig. 4.30 C3A細胞培養於丙烯酸接枝後纖維連接蛋白塗布 98

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