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研究生： 魏俐德
Wei, Li-Te
論文名稱： 探討循環拉伸與氧化壓力於黃韌帶細胞之關係
Investigating the Relationship Between Cyclic Stretching and Oxidative Stress in Ligamentum Flavum Cells
指導教授： 涂庭源
Tu, Ting-Yuan
共同指導教授: 林政立
Lin, Cheng-Li
學位類別： 碩士
Master
系所名稱： 工學院 - 生物醫學工程學系
Department of BioMedical Engineering
論文出版年： 2021
畢業學年度： 109
語文別： 英文
論文頁數： 47
中文關鍵詞： 黃韌帶肥厚 、循環拉伸 、細胞排列 、氧化壓力 、纖維化
外文關鍵詞： ligamentum flavum hypertrophy, cyclical stretch, cell alignment, oxidative stress, fibrosis
相關次數： 點閱：188  下載：0 
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    • 腰椎管狹窄症(LSCS)是常見於老年人身上的脊椎疾病之一，黃韌帶肥厚則是造成LSCS的主要原因之一。黃韌帶是位於腰椎管背側的韌帶結構，可以保護腰椎管內的神經，一旦肥厚便會壓迫到神經並造成LSCS。
    目前對於黃韌帶肥厚的原因還沒有很完整的研究，但是從病人組織的觀察及一些動物實驗上了解到機械應力似乎是造成黃韌帶肥厚的主因，並且黃韌帶肥厚的組織有一些共同特徵，例如纖維化及細胞外基質組成的改變。
    但是僅透過組織的觀察及動物實驗無法詳細的了解黃韌帶細胞逐漸肥厚的進程，因此體外的細胞實驗對於研究黃韌帶肥厚的機制也是相當重要的。
    在本研究中從黃韌帶肥厚的病人組織中培養細胞進行研究，並在二維及三維的環境下給予單軸的循環拉伸力。實驗後發現單軸的循環拉伸會影響細胞的排列，並且添加抗氧化劑後會減緩拉伸力對細胞排列的影響;另外，也發現循環拉伸確實導致細胞內氧化壓力的增加並且影響細胞外基質的表現。但是最終嘗試了解黃韌帶細胞被施加機械應力後是以什麼纖維化途徑導致最終細胞外基質的組成改變時，卻沒有得到確切的結果。
    在這個研究中成功的觀察到施加機械應力後，細胞隨時間改變有不同的反應，是與體內實驗的不同之處。在未來也希望透過更多的實驗了解更詳細的導致黃韌帶肥厚的機制。

    Lumbar spinal canal stenosis (LSCS) is one of the common spine diseases in the elderly, and ligamentum flavum hypertrophy (LFH) is one of the main causes of LSCS. The ligamentum flavum (LF) is a ligament structure located on the dorsal side of the lumbar spinal canal, which can protect the nerves in the lumbar spinal canal. Once it is hypertrophy, it will compress the nerves and cause LSCS.
    At present, there is no complete research on the causes of LFH; however it is noticed from patient tissue observations and some animal experiments that the mechanical stress seems to be the main cause of LFH, and the tissues of LFH have some common features, such as fibrosis and changes in the composition of the extracellular matrix (ECM). Nevertheless, it is impossible to observe the process of LF cell hypertrophy in detail through tissue observation and animal experiments. Therefore, in vitro cell experiments are also very important for studying the mechanism of LFH.
    In this study, cells were cultured from patient tissues with LFH, and uniaxial cyclic stretching was given in a 2D and 3D environment. Experiments have found that uniaxial cyclic stretching will affect the alignment of cells, and the addition of antioxidants will slow down the effect of stretching on the cell alignment; in addition, the cyclic stretching does cause an increase in oxidative stress in the cell and affects the expression of ECM of the cell. However, when trying to understand the fibrosis pathway caused the change of composition of the ECM on the LF cells after mechanical stress, no evidence was obtained.
    After applying mechanical stress, different responses on LF cell over times were successfully observed that is different from in vitro experiments, In the future, it is hoped to learn more about the mechanism that causes LFH through more experiments.

    摘要 I Abstract II 致謝 IV Contents V List of Table VII List of Figures VIII Chapter 1. Introduction 1 1.1 Background 1 1.1.1 Introduction of ligamentum flavum 1 1.1.2 Previous research on LFH 2 1.2 Literature reviews 4 1.2.1 Two-dimensional (2D) cyclic stretching increases the reactive oxygen species (ROS) of LF cells 4 1.2.2 2D cyclic stretching affects collagen secreted of LF cells 4 1.2.3 2D cyclic stretching affects cell alignment 5 1.3 Motivation and specific aims 5 Chapter 2. Materials and Methods 7 2.1 Experimental process 7 2.1.1 2D stretching experiment 7 2.1.2 3D stretching experiment 8 2.2 Cell culture 9 2.2.1 LF cell culture 9 2.2.2 Cell counts 10 2.2.3 Antioxidant treatment 11 2.3 Mechanical stretching device and cyclic stretching membrane 11 2.3.1 Mechanical stretching device 11 2.3.2 2D cyclic membrane and surface modification 12 2.3.3 Preparation of 3D PDMS membrane 13 2.3.4 Surface modification of 3D stretching membrane 15 2.4 3D collagen-elastin matrix preparation 17 2.5 Live/Dead cell staining 19 2.6 DCFDA / H2DCFDA fluorescent staining and measuring 20 2.7 Western blot 21 2.8 Image quantization 23 2.8.1 LF cells viability 23 2.8.2 LF cells alignment 24 2.8.3 Reactive oxygen species (ROS) quantification 24 2.8.4 Protein quantification 24 2.9 Statistical analysis 24 Chapter 3. Results and Discussion 26 3.1 2D cyclic stretching experiments 26 3.1.1 The viability of LF cells under different stretching conditions 26 3.1.2 Cell alignment under different stretching conditions 27 3.1.3 The cell alignment under an extreme stretching and NAC added condition 28 3.1.4 Quantification of ROS under extreme stretching and NAC added conditions 30 3.1.5 Quantification of fibrotic proteins 32 3.1.6 Quantification of ECM proteins 34 3.2 3D cyclic stretching experiments 36 3.2.1 The viability of LF cells 36 3.2.2 Quantification of ROS 37 3.2.3 Antioxidant inhibits oxidative stress 39 3.2.4 Quantification of ECM protein 41 Chapter 4. Conclusion 43 Chapter 5. Future works 44 Reference 45

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