製備具熱敏性之可拉伸導電薄膜以應用 於肌肉組織工程

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姓名

鍾永智(Yong-Zhi Zhong) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

製備具熱敏性之可拉伸導電薄膜以應用於肌肉組織工程
(Preparation of Stretchable Conductive Films with Thermosensitivity for Muscle Tissue Engineering Application)

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摘要(中)

機械和電訊號對肌肉生成有正向作用。在我們先前研究中成功地以聚二甲基矽氧烷(PDMS)薄膜作為彈性基底將導電高分子聚吡咯(PPy)製備於其上得到高拉伸性導電薄膜。可用於組裝生物反應器對細胞進行拉伸及電刺激。然而，如何將刺激過後的細胞完整取下來應用於後續肌肉修復是一大挑戰。因此本研究將製備同時具有導電性及溫敏性的薄膜，並以小鼠骨骼成肌細胞C2C12作為研究對象，研究所開發薄膜是否合適對貼附的細胞進行拉伸跟電刺激，並探討降溫細胞脫附後細胞外基質及肌管排列是否得以保留。在電刺激的部份我們發現此薄膜上的細胞在1 V/cm的電場刺激下有最好效果。在細胞排列實驗中9%循環拉伸促使C2C12沿著與拉伸方向垂直的方向進行高度排列。而複合刺激實驗中其中細胞排列取決於拉伸方向，可得到高度分化且排列的肌管。在西方點墨的實驗中，證實利用降溫得到的細胞片相較於傳統胰蛋白酶消化細胞方法仍保有細胞外基質。qPCR結果證實拉伸刺激或電刺激皆能夠增加分化標誌基因的表達，肌球重鏈蛋白(MHC)免疫染色實驗指出兩種刺激都會有效地增加肌管的數量及提升分化率，且拉伸刺激有助於控制肌管的排列。與單一刺激相比，複合刺激不僅保有維持肌管排列的效果，還能使分化標誌基因更加上調控或維持高水準，並在降溫脫附後仍保留這些排列的肌管。這些結果證實本研究所開發的可助於收集拉伸及電刺激過後的細胞片，以作為組織工程及再生醫學上的應用。

摘要(英)

In our previous study, our lab has successfully deposited conductive polypyrrole (PPy) onto elastic polydimethylsiloxane (PDMS) substrate to obtain a highly stretchable conductive film. It can be used to construct a bioreactor to cyclicaly stretch and electrically stimulate surface cells. However, how to completely harvest these stimulated muscle tissue to repair damage muscle is a challenge. Therefore, in this study, we intend to develop a polymer film owns both conductivity and thermo-sensitivity. Therefore, cells after stimulation can be completely harvested as cell sheets through breducing temperature. Mouse skeletal myoblast, C2C12 cells, were applied to examine our hypothesis. In electrical stimulation, C2C12 cells demonstrated the best myo-differentiation under the electric field of 1 V/cm. Regarding cyclic stretching, the strain equal to or higher than 9% can highly align C2C12 perpendicular to the stretching direction. The Western blotting experiments demonstrated that the cell sheets harvested by cooling reserved more extracellular matrix (ECM) than cells collected by the traditional trypsin digestion method. The qPCR results revealed that both cyclic stretching and electrical treatment increased the expression of differentiation myogenic marker genes. Immunostaining of myosin heavy chain protein (MHC) indicated that both mechanical and electrical stimuli effectively increased the number of myotubes and the differentiation ratio, and the myotubes can be aligned by the cyclic stretching. Compared the sole stimulation, dual stimulation not only aligned myotubes, but also highly increased the up-regulation myogenic differentiation markers. Stimulated cell sheets can be harvested by cooling and the alignment of myotubes was still maintained. These results suggested that our developed polymeric film can be applied to harvest intact cell sheets after cyclic stretching and electrical stimulation, which increased the feasibility bioreactor for the application of tissue engineering and regenerative medicine.

關鍵字(中)

★ 熱敏
★ 導電
★ 機電刺激
★ 細胞

關鍵字(英)

論文目次

摘要 III
Abstract VI
目錄 VII
表目錄 XII
第一章緒論 1
1-1 研究背景 1
1-2 研究目的 2
第二章文獻回顧 4
2-1 細胞片工程 4
2-2 細胞片工程應用於心肌缺血修復 9
2-3 細胞片工程應用於心肌缺血修復 10
2-4 細胞片工程應用於血管修復 11
2-5 細胞片工程應用於膀胱修復 11
2-6 熱敏高分子 12
2-6-1熱敏高分子聚合 13
2-6-2熱敏高分子與彈性分子結合 14
2-6-3熱敏高分子與導電性分子結合 15
2-7生物刺激細胞收穫裝置 15
第三章實驗藥品、儀器及方法 18
3-1 實驗藥品 18
3-1-1 材料製備藥品 18
3-1-2 生物實驗藥品 19
3-2 實驗儀器 25
3-3 實驗方法 27
3-3-1 設計、組裝生物刺激裝置 27
3-3-2 電阻量測 34
3-3-3 細胞培養、繼代、冷凍及解凍 35
3-3-4 分化血清配製及肌管分化 37
3-3-5 循環拉伸對肌管分化的影響 38
3-3-6 電刺激對肌管分化的影響 41
3-3-7 拉伸/電複合刺激對肌管分化的影響 42
3-3-8 膜對生物活性之影響 44
3-3-9 MHC免疫螢光染色及分析 46
3-3-10 細胞排列分析 48
3-3-11 即時聚合酶反應儀 (Real-time PCR) 49
3-3-12 細胞脫附 57
3-3-13 西方點墨 (Western blot) 57
第四章結果與討論 60
4-1 材料性質 60
4-1-1 ATR-FTIR分析 60
4-1-2 XPS分析 62
4-1-3 SEM 分析 65
4-1-4 表面接觸角測量 66
4-1-5 薄膜透光度分析 68
4-2 拉伸和電阻測試 70
4-2-1 裝置電阻之測試 70
4-2-2 裝置通電阻之溫度量測 72
4-3生物活性分析 73
4-3-1 MTT分析 73
4-3-2 拉伸對C2C12排列之影響 75
4-4-3 循環拉伸對C2C12分化之影響 79
4-4-4 電刺激對C2C12分化之影響 82
4-4-5 複合刺激對C2C12分化之影響 84
5-1細胞脫附 96
5-1-1 細胞脫附 96
5-1-2 細胞免疫螢光染色 100
5-1-3 細胞片蛋白質分析 100
5-1-4 細胞片Live /Dead 分析 101
結論 103
參考文獻 105

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指導教授

胡威文(Wei-Wen Hu)

審核日期

2020-8-24

推文