搭建可提供電刺激與機械刺激之生物反應器

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姓名

楊士永(Shih-Yung Yang) 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

搭建可提供電刺激與機械刺激之生物反應器
(Fabrication of a bioreactor for applying mechanical and electrical stimulations)

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摘要(中)

電刺激與機械刺激在組織工程中扮演著極為重要的角色，為了研究他們對於細胞及組織的影響，開發一能夠同時進行電刺激及機械刺激的生物反應器是必要的。在這項研究中，我們透過將聚?咯（PPy）聚合於二甲基矽氧烷（PDMS）薄膜上，製作了一可拉伸的導電薄膜。為了促進PPy的聚合，我們用氫氧化鈉(NaOH)水溶液對PDMS薄膜進行預處理，修飾其表面以增加粗糙度。NaOH的預處理時間直接影響了PPy/PDMS薄膜的電性質以及生物適合性。在對其電性質可靠度進行測試後，確定其在拉伸30~40%應變量之間仍可保有低且穩定的電阻值(20kΩ)。接著透過MTT測試證實導電薄膜有助於表面細胞之活性。綜合電性質與MTT之結果，我們發現經過NaOH預處理6小時之PPy/PDMS薄膜具有最穩定之電性質與生物相容性。該薄膜雖然外觀呈現黑色，但其具有透光性質，因此可以透過顯微鏡直接觀察培養於上方的細胞。將PPy/PDMS薄膜結合流道層、培養層，製造出能夠對細胞進行電刺激以及機械刺激之生物反應器並且將其投入實測中，首先對C2C12進行頻率為0.5 Hz且應變量為10%之循環拉伸刺激，由結果可以看到循環拉伸可以有效的改變細胞的排列方向，在肌管分化實驗結果顯示循環拉伸可以有效控制肌管的排列同時也有效提升肌管的分化能力，並且對肌管分化的指標基因如肌細胞生成素(Myogenin)和肌球蛋白重鍊(myosin heavy chain,MHC)有上調控的效果。另外在電刺激的部份，則是以0.1、0.33及1V/cm的直流電進行刺激，結果顯示雖然電刺激對於細胞的排列並無顯著的影響，但可以有效提升肌管的分化效率。最後由qPCR的結果顯示，直流電刺激雖然造成MyoD的基因表現被抑制，但對於肌管分化的指標基因Myogenin和MHC都有顯著上升的調控。以上實驗結果，證實所開發的生物反應器可以對C2C12細胞進行循環拉伸及電刺激，藉以促進其分化能力。未來該裝置除了能進行肌肉細胞體外刺激培養，也可以用於研究肌肉相關疾病對物理刺激的反應機轉。

摘要(英)

Electrical and mechanical stimulations both play important roles in the tissue engineering. In order to simultaneously explore their effects on tissue regeneration, it is essential to develop a bioreactor to electrically and mechanically stimulate cells at the same time. In this study, we fabricated a stretchable conductive film by polymerizing pyrrole on PDMS surface. To promote the deposition of polypyrrole (PPy), PDMS substrates were pretreated NaOH solution to increase surface roughness, and this pretreatment was critical to the electrical property and biocompatibility of PPy/PDMS films. The prepared PPy/PDMS films demonstrate excellent electrical properties that they maintained low electic resistance (20kΩ) under 30~40% tensile strain and were reliable during 200 cycles of 10% strain. The results MTT assay revealed that these conductive PPy/PDMS films highly promoted viability of C2C12 cells. We pretreated PDMS NaOH solution for 6 hours in the following experiment because this group demonstrated best electrical properties and biocompatibility. These PPy/PDMS films were dark but transparent and thus can be easily applied for monitoring surface via the microscope. Therefore, these PPy/PDMS films were applied to build a bioreactor for the application of electrical and mechanical stimulations. Firstly, this device was applied for mechanical stimulation that cells were cyclically stretched for 6 hours of 10 % tensile strain at 0.5Hz for 4 days. The results showed that the treated cells were aligned perpendicular to the stretching direction. The result myotube differentiation suggested that cyclic stretching effectively not only aligned myotubes perpendicular to the stretching direction but also improved the index of differentiation. Moreover, genes correlative to myotube differentiation including myogenin and myosin heavy chain (MHC), were also promoted. Regarding to electrical stimulation, 0.1, 0.33, and 1V/cm were applied to treat surface cells for 4 hour each day for 4 days. Although there was no significant effect on the cell alignment, the MHC expression and differentiation index were highly improved. The results of qPCR showed that both myogenin and MHC genes were upregulated, but the MyoD has been inhibited. These results suggested that our developed bioreactor is feasible to mechanically and electrically stimulate C2C12 cells to promote myogenic differentiation. This device can be applied not only promote muscle tissue regeneration in vitro but also as a model to investigate the pathology muscle disease.

關鍵字(中)

★ 電刺激
★ 機械刺激
★ 生物反應器
★ 小鼠肌肉纖維母細胞

關鍵字(英)

★ electrical stimulation
★ mechanical stimulation
★ bioreactor
★ C2C12

論文目次

摘要 I
Abstract III
目錄 VI
圖目錄 VIII
表目錄 XI
第一章緒論 1
1-1 前言 1
第二章文獻回顧與理論基礎 3
2-1 組織工程 3
2-1-1 骨骼肌組織工程 5
2-2 生醫材料 7
2-2-1 導電高分子 8
2-3 肌肉組織 9
2-3-1 肌肉的組成 9
2-3-2 肌肉的發生過程 11
2-4 機械刺激對肌肉細胞的影響 12
2-4-1 機械刺激裝置 16
2-5 電刺激對肌肉的影響 19
2-6 結合機械刺激與電刺激對肌肉的影響 22
第三章實驗方法及設備 24
3-1 實驗藥品 24
3-1-1 材料製備藥品 24
3-1-2 細胞培養及肌肉分化藥品 26
3-1-3 免疫螢光染色用藥品 28
3-1-4 RNA萃取、反轉錄cDNA、PCR試劑 29
3-1-5 其他藥品 30
3-2 實驗儀器 30
3-3 試藥製備及實驗方法 32
3-3-1 生物反應器設計、製作 32
3-3-2 微接觸印刷（Micro contact printing） 41
3-3-3 細胞繼代培養與與冷凍解凍 45
3-3-4 肌管分化培養液配方 47
3-3-5 肌管分化 47
3-3-6 免疫螢光染色 48
3-3-7 細胞骨架染色 49
3-3-8 螢光染色分析 50
3-3-9 電性質量測 50
3-3-10 MTT生物活性分析 51
3-3-11 即時聚合?反應儀 ( qPCR ) 53
3-3-12 掃描式電子顯微鏡 56
3-4 實驗設計與架構 57
3-4-1 材料之物理性質 57
3-4-2 材料之化學定性 57
3-4-2 材料之生物適合性 57
3-4-3 物理性刺激對細胞型態及排列的影響 57
3-4-4 物理性刺激對肌管分化的影響 58
第四章實驗結果與討論 59
4-1 材料之性質 59
4-1-1 表面特徵 59
4-1-2 親疏水性 62
4-1-3 紅外線光譜FTIR-ATR分析 64
4-1-4 電性質 68
4-1-5 機械應變對材料電性質之影響 69
4-1-6 細胞培養之光學成像 71
4-2 材料之生物適合性測定 72
4-2-1 MTT 測試對細胞活性分析 72
4-3 微接觸印刷 75
4-4 循環拉伸對細胞之影響 76
4-5 循環拉伸對肌管分化之影響 80
4-5-1 循環拉伸對C2C12肌管分化與排列之影響 80
4-5-2往復式循環拉伸對C2C12肌管分化調控基因的影響 83
4.6 直接電刺激對細胞之影響 86
4-6-1 直接電刺激對細胞活性及排列之影響 86
4-6-2直接電刺激對C2C12肌管分化的影響 89
結論 94
參考文獻 96

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指導教授

胡威文(Wei-Wen Hu)

審核日期

2018-8-24

推文