臺灣博碩士論文加值系統

摘要
導電高分子同時具有有機高分子的物理和化學特性以及金屬的導電性，已被應用在光電材料及生物感測上。本研究旨在探討以電紡方式所製備的聚鄰甲氧基苯胺奈米絲薄膜之生物相容性及其在組織工程應用之可行性。掃瞄式電子顯微鏡結果發現聚鄰甲氧基苯胺奈米絲直徑在 200 ~ 300 nm；表面接觸角為 104 ± 8¬ o，為疏水性結構；熱重分析儀分析得知其裂解溫度在300 ~ 600 oC；循環伏安法分析得知本材料具有氧化還原之特性。根據 ISO 10993-5 的規範，我們發現聚鄰甲氧基苯胺奈米絲薄膜的間接細胞毒性，不論是在電刺激前後，皆屬於生物體可接受的範圍。L929 小鼠纖維母細胞、C2C12 小鼠肌原母細胞之貼附與增生結果發現，聚鄰甲氧基苯胺奈米絲薄膜對於C2C12細胞的貼附以及增生均較L929細胞來的佳；進一步探討C2C12 細胞之MyoD、Myogenin、Myf-5、MRF4基因表現，初步證實聚鄰甲氧基苯胺奈米絲薄膜可促進 C2C12 細胞之分化能力。利用巨噬細胞探討材料之體外生物相容性，發現聚鄰甲氧基苯胺奈米絲薄膜會誘發巨噬細胞釋放些微一氧化氮與發炎激素；進ㄧ步量測 LDH 的釋放量證明材料對細胞會造成些微損傷，引起些微發炎反應，但仍屬可接受之範圍。蛋白質吸附試驗發現，此材料在與人類血清白蛋白以及血漿纖維蛋白原反應 48 小時之吸附量達 80 μg/cm2。將聚鄰甲氧基苯胺奈米絲薄膜植入 Wistar 大鼠背部皮下，ㄧ及四週之組織切片中發現有些許嗜中性球、巨噬細胞的聚集，八及十二週則有血管的增生，證實聚鄰甲氧基苯胺奈米絲薄膜不會引起慢性發炎反應。本研究初步證實聚甲氧基苯胺電紡絲薄膜屬於低毒性、良好生物相容性、低蛋白質吸附、低免疫抗原性；本材料雖然不利於纖維母細胞貼附，但卻適合肌肉細胞之貼附生長，此材料未來可能適合應用於肌肉組織工程。

Abstract
Conducting polymers have both the physical and chemical characteristics of organic polymer as well as the conductivity of metals, which make them suitable to be used as photoelectric or biosensing materials. The purpose of the research was to study the biocompatibility of electrospinning poly o-methylaniline nanofiber and the feasibility for tissue engineering application. According to the SEM images, the diameter of poly o-methylaniline nanofiber was between 200 and 300 nm. Contact angle was 104 ± 8¬ o, which made its surface hydrophobic. Thermogravimetry analysis revealed that Tg was between 300 and 600 oC. The material had redox potential by cyclic voltammetry analysis. According to the ISO 10993-5 specification, we found that cytotoxicity of electrospinning poly o-methylaniline nanofiber was under the scope of acceptance prior and after electrical stimulation. From cell attachment and proliferation study, L929 fibroblasts had poor cell adhesion and proliferation on electrospinning poly o-methylaniline nanofiber compared with C2C12 myoblasts. By studying the gene expression of myoD, myogenin, myf-5, MRF4, myogenic differentiation of C2C12 cells was enhanced. In vitro biocompatibility studied by macrophages found that electrospinning poly o-methylaniline nanofiber induced the release of nitric oxide and inflammation cytokines. LDH release further demonstrated that the material caused some cell damage which elicited minor inflammation. Protein adsorption assay showed that human serum albumin and human plasma fibrinogen reached 80 μg/cm2 after 48 hr. In vivo biocompatibility was performed by implanting electrospinning poly o-methylaniline nanofiber in the back of Wistar rats subcutaneously. Some neutrophil and macrophages appeared after 1 and 4 weeks, and blood vessels were observed after 8 and 12 weeks. Our study suggested that electrospinning poly o-methylaniline nanofiber had relatively low toxicity, good biocompatibility, low protein adsorption and low immunogenicity. Although the material may not be suitable for fibroblast attachment, it is suitable for the attachment and growth of myoblasts. Electrospinning poly o-methylaniline nanofiber may be applied in muscle tissue engineering applications.

目錄
摘要 I
目錄 IV
圖索引 X
縮寫表 XIII
第一章 緒論 1
1.1前言 1
1.2 理論基礎 2
1.2.1 聚苯胺特性簡介 2
1.2.2 聚苯胺高分子不同之合成方法 3
1.2.3 電紡絲之簡介 4
1.2.4 聚苯胺之生物相容性及其在組織工程上之應用 5
1.2.5 免疫細胞激素 6
1.2.6 肌原母細胞分化調節因子 10
1.3 文獻回顧 11
1.3.1生物材料於組織工程上之應用 11
1.3.2 其他導電高分子相關之應用 12
1.3.3 靜電纺絲所製備之材料生物相容性探討 12
1.4 研究動機與目的 15
第二章 材料與方法 16
2.1.研究架構 16
2.2材料製備 18
2.2.1 聚鄰甲氧基苯胺聚合 18
2.2.1.1 溶劑製備 18
2.2.1.2 聚鄰甲氧基苯胺(poly o-methoxyaniline)高分子合成 18
2.2.2 聚鄰甲氧基苯胺電紡製備 19
2.2.3 聚鄰甲氧基苯胺電紡絲電刺激 21
2.2.3.1電刺激實驗裝置製備 21
2.2.3.2材料電刺激 21
2.3藥品配製 23
2.3.1 小鼠 L929 纖維母細胞培養基 α-MEM 配製 23
2.3.2 人類 U-937 淋巴瘤細胞培養基RPMI 1640配製 23
2.3.3 小鼠 RAW 264.7 巨噬細胞培養基 DMEM 配製 24
2.3.4 小鼠 C2C12 肌原母細胞培養基 DMEM 配製 24
2.3.5小鼠 C2C12 肌原母細胞分化培養基 DMEM 配製 25
2.3.6 磷酸鹽緩衝溶液配製 25
2.3.7小鼠 RAW 264.7 巨噬細胞磷酸鹽緩衝溶液配製 26
2.3.8 胰蛋白酶配製 27
2.3.9 Trypan Blue 染劑配製 27
2.3.10 脂多醣體配製 27
2.3.11 佛波肉荳蔻醋酸配製 27
2.3.12 中性紅配製 27
2.3.13 Propidium Iodide 配製 28
2.3.14 Fluorescein diacetate 配製 28
2.3.15 人類血清白蛋白配製 28
2.3.16 人類血將纖維蛋白原配製 28
2.3.17 十二烷基硫酸鈉配製 28
2.3.18 乳酸脫氫酶測試溶液配製 28
2.3.19 Triton X-100 溶液配製 29
2.4 細胞來源與細胞培養操作技術 29
2.4.1 細胞來源及繼代培養 29
2.4.1.1小鼠 L929 纖維母細胞 29
2.4.1.2 人類 U-937 淋巴瘤細胞 30
2.4.1.3 小鼠 RAW 264.7 巨噬細胞 31
2.4.1.4小鼠 C2C12 肌原母細胞 32
2.4.2 細胞凍存 35
2.4.3 細胞解凍 35
2.5 細胞計數 36
2.6 掃描式電子顯微鏡觀察 36
2.6.1 SEM樣品製備方法 36
2.7熱重分析試驗 37
2.8電化學分析試驗 38
2.9 接觸角試驗 39
2.10 瓊脂擴散試驗 41
2.10.1 試驗方法 41
2.11 細胞貼附試驗 43
2.12 細胞增生試驗 44
2.13 乳酸脫氫酶釋放量分析 45
2.14 蛋白質吸附試驗 46
2.14.1蛋白質定量試驗 47
2.15 一氧化氮試驗 48
2.15.1 試驗方法 49
2.16 細胞激素釋放量分析 51
2.16.1 腫瘤壞死因子-α釋放量分析 51
2.16.1.1 試驗方法 51
2.16.2 細胞介白素-1β 釋放量分析 52
2.16.2.1 試驗方法 52
2.16.3 細胞介白素-6 釋放量分析 53
2.16.3.1 試驗方法 53
2.16.4 環氧化酶-2 釋放量分析 54
2.16.4.1 試驗方法 54
2.17 組織切片 55
2.17.1 組織前處理 56
2.17.2 脫水 56
2.17.3 臘塊包埋 57
2.17.4 切片 57
2.17.5 染色 58
2.18 反轉錄聚合酶反應 59
2.18.1 Primer 設計 60
2.18.2 RNA 萃取及純化 61
2.18.3 反轉錄作用 62
2.18.4 聚合酶連鎖反應 62
2.18.5 DNA瓊膠電泳 62
2.19 劉氏染色法 63
2.20 統計分析 63
第三章 結果 64
3.1 材料特性分析 64
3.1.1掃描式電子顯微鏡觀察 64
3.1.2熱重分析試驗 66
3.1.3電化學分析試驗 68
3.1.4接觸角試驗 70
3.2 細胞存活率分析 73
3.2.1瓊脂擴散間接毒性試驗 73
3.2.2 細胞貼附試驗 77
3.2.2.1 小鼠 L929 纖維母細胞貼附試驗 77
3.2.2.2 小鼠 C2C12 肌原母細胞貼附試驗 80
3.2.3 細胞增生試驗 83
3.2.3.1 小鼠 L929 纖維母細胞增生試驗 83
3.2.3.1小鼠 C2C12 肌原母細胞增生試驗 86
3.2.4乳酸脫氫酶釋放量分析 89
3.3蛋白質吸附試驗分析 90
3.4免疫反應測定 92
3.4.1一氧化氮釋放量測定 92
3.4.2 細胞激素釋放量測定 94
3.4.2.1 腫瘤壞死因子-α釋放量分析 94
4.4.2.2 細胞介白素-1β 釋放量分析 94
4.4.2.3 細胞介白素-6 釋放量分析 95
4.4.2.4 環氧化酶-2 釋放量分析 95
3.4.3 組織切片 100
3.4.3 肌原母細胞分化基因表現 104
第四章 討論 106
4.1 聚鄰甲氧基苯胺電紡絲薄膜材料特性之探討 107
4.2 聚鄰甲氧基苯胺電紡絲薄膜細胞毒性之探討 109
4.3 聚鄰甲氧基苯胺電紡絲薄膜對於蛋白質吸附之特性探討 111
4.4 聚鄰甲氧基苯胺電紡絲薄膜對於免疫反應之探討 112
4.5 聚鄰甲氧基苯胺電紡絲薄膜對肌原母細胞基因表現探討 115
第五章 結論與未來展望 116
參考文獻 118
附錄表 128
A.儀器表 128
B.藥品資料表 131

圖索引
圖 2-1研究架構 17
圖 2-2鄰甲氧基苯胺單體 18
圖 2-3聚苯胺示意圖 19
圖 2-4 電紡絲儀器架設示意圖 20
圖 2-5聚鄰甲氧基苯胺電紡絲薄膜 20
圖 2-6 電刺激實驗裝置示意圖 22
圖 2-7 細胞型態圖 33
圖 2-8 循環伏安法電位掃描 39
圖 2-9 接觸角測量示意圖 40
圖 2-10材料細胞毒性判斷方式之示意圖 42
圖 2-11 ㄧ氧化氮分析示意圖 50
圖 3-1聚鄰甲氧基苯胺電紡絲薄膜掃描式電子顯微鏡影像拍攝………..…..65
圖 3-2 聚鄰甲氧基苯胺電紡絲薄膜熱重分析儀曲線圖 67
圖 3-3聚鄰甲氧基苯胺電紡絲薄膜循環伏安法分析曲線圖 69
圖 3-4 聚鄰甲氧基苯胺電紡絲薄膜接觸角分析圖 72
圖 3-5 顯微鏡拍攝瓊脂擴散試驗細胞生長情況 75
圖 3-6 小鼠 L929 細胞貼附於材料上方之FDA/PI螢光染色影像 78
圖 3-7小鼠L929細胞於聚鄰甲氧基苯胺電紡絲薄膜之貼附情形 79
圖 3-8小鼠 C2C12 細胞貼附於材料上方之FDA/PI螢光染色影像 81
圖 3-9 小鼠C2C12 細胞於聚鄰甲氧基苯胺電紡絲薄膜之貼附情形 82
圖 3-10小鼠 L929 細胞於材料上方之FDA/PI 增生螢光染色影像 84
圖 3-11小鼠 L929 細胞於聚鄰甲氧基苯胺電紡絲薄膜之增生情形 85
圖 3-12小鼠 C2C12 細胞於材料上方之FDA/PI 增生螢光染色影像 87
圖 3-13 小鼠 C2C12 細胞於聚鄰甲氧基苯胺電紡絲薄膜之增生情形 88
圖 3-14 小鼠 RAW 264.7 巨噬細胞與材料共培養後 LDH 釋放結果 89
圖 3-15 聚鄰甲氧基苯胺電紡絲薄膜之蛋白質吸附情形 91
圖 3-16 小鼠 RAW 264.7 巨噬細胞與材料共培養後ㄧ氧化氮分泌量 93
圖 3-17 巨噬細胞與材料共培養後腫瘤壞死因子-Α釋放圖 96
圖 3-18 巨噬細胞與材料共培養後細胞介白素-1Β釋放圖 97
圖 3-19 巨噬細胞與材料共培養後細胞介白素-6 釋放圖 98
圖 3-20 巨噬細胞與材料共培養後環氧化酶- 2 釋放圖 99
圖 3-21 利用超音波探測材料植入小鼠皮下部位圖 100
圖 3-22 將聚鄰甲氧基苯胺電紡絲植入小鼠皮下之組織切片圖 103
圖 3-23 材料對於小鼠 C2C12 細胞之分化、基因表現 106

表索引
表 1-1 細胞激素釋放之作用功能及其來源 9
表 2-1 細胞培養方式 34
表 2-2 瓊脂擴散之細胞毒性測試範圍描述定義 43
表 2-3 BCA 標準曲線之白蛋白濃度配製表 48
表 2-4 本研究所使用之引子序列及 PCR 反應條件 60
表 3-1 聚鄰甲氧基苯胺電紡絲薄膜熱重分析儀數據表...………...………...67
表 3-2 瓊脂擴散試驗之指數 76
表 3-3 瓊脂擴散試驗結果 76

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