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研究生： 楊睿濬
Yang, Ruei-Jyun
論文名稱： 研究黃韌帶TSP-1/TGF-β1 信號通路和氧化壓力的肥厚治療潛力
Investigating the Therapeutic Potential of Targeting TSP-1/TGF-β1 Signaling Pathways and Oxidative Stress in the Management of Ligamentum Flavum Hypertrophy
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： 2023
畢業學年度： 111
語文別： 英文
論文頁數： 48
中文關鍵詞： 黃韌帶 、黃韌帶肥厚 、老化 、缺氧 、纖維化
外文關鍵詞： ligamentum flavum, LF hypertrophy, aging, hypoxia, fibrosis
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    • 腰椎管狹窄症(LSCS)是種常見的退行性疾病，影響人數受醫療進步和社會高齡化逐漸增加，會受腰痛、間歇性跛行而影響患者活動能力。發病原因主要黃韌帶(LF)所致，在長期機械應力下炎症將導致黃韌帶不斷修復進而疤痕增大形成黃韌帶肥厚(LFH)。當組織受損時修復機制會發生大量的轉化生長因子1(TGF-β1)和活性氧物質(ROS)的產生以及傷口附近會發生血管新生。先前研究表明老化細胞會有缺氧現象以及其對環境刺激的反應大幅下降，恢復能力降低。組織過度地修復產生的疤痕堆積造成纖維化，其中轉化生長因子1參與機制最為重要。而血小板和成纖維細胞所分泌的血小板反應蛋1(TSP-1)的眾多作用中有著激活潛在轉化生長因子1去進行纖維化的功能。過去文獻中並沒有研究血小板反應蛋1在黃韌帶所扮演的角色，本研究旨在探討血小板反應蛋1如何對轉化生長因子1和活性氧物質去影響纖維化以及藥物LSKL作為治療黃韌帶肥厚的潛力。首先在黃韌帶肥厚的病患的組織切片觀察蛋白表達，在體外培養黃韌帶細胞加入FG-4592模擬缺氧環境。結果表明在病理狀態的黃韌帶有著轉化生長因子1和血小板反應蛋1高表達並且缺氧環境的ROS和血小板反應蛋1水平也提高，証實黃韌帶肥厚裡有這些因子的參與。被潛在轉化生長因子1和血小板反應蛋1所處理過的細胞也有兩者相依性的表達，纖維化程度被調控。透過活性氧物質的抑制劑NAC以及LSKL一種阻止血小板反應蛋1去激活潛在轉化生長因子1的藥物去探討治療黃韌帶纖維化的成效。本研究可以為黃韌帶肥厚提出新的致病因子以及未來預防或是治療的方法。

    Lumbar spinal canal stenosis (LSCS) increasingly affects people due to medical advances and an aging population. It causes lumbar pain and intermittent claudication, impacting mobility. LSCS primarily arises from ligamentum flavum (LF) thickening (LFH) due to chronic mechanical stress and inflammation. Tissue damage triggers transforming growth factor-beta 1 (TGF-β1) and reactive oxygen species (ROS) production, along with neovascularization as the repair mechanism. Aging cells experience hypoxia and reduced responsiveness, leading to diminished regenerative capacity. Excessive tissue repair leads to fibrosis, with TGF-β1 playing a crucial role. Thrombospondin-1 (TSP-1) activates latent TGF-β1 to induce fibrosis. However, TSP-1's role in LF remains unstudied. This research investigates TSP-1's influence on TGF-β1 and ROS-mediated fibrosis, along with LSKL as a treatment for LF hypertrophy. Protein expression in LF tissue slices from hypertrophy patients was observed. In vitro, experiments simulate hypoxic conditions using FG-4592. The results confirm elevated expression of TGF-β1 and TSP-1 in pathologic LF, along with increased ROS and TSP-1 under hypoxia. Cells treated with latent TGF-β1 and TSP-1 show dependent expression increase, regulating fibrosis. The efficacy of NAC (a ROS inhibitor) and LSKL (a TSP-1-blocking drug) for LF fibrosis treatment ww explored. This study provides insights into LF hypertrophy's pathogenic factors and potential treatment approaches.

    摘要 i Abstract ii 致謝 iii Contents iv List of Figures vi List of Abbreviations x Chapter 1 Introduction 1 1.1 Background 1 1.2 Fibrosis 2 1.2.1 Fibroblast 2 1.2.2 Hypoxia in LFH patient 4 1.2.3 TSP-1 5 1.2.4 TGF-β1 6 1.2.5 ROS 8 1.3 Aims of the research 8 Chapter 2 Materials and Methods 10 2.1 Experimental workflows 10 2.2 Materials 11 2.3 Recipes 13 2.4 LF tissue 14 2.5 Immunohistochemistry (IHC) staining 15 2.6 Cell culture 16 2.7 Cell counting kit-8 (CCK-8) 17 2.8 Western blot analysis 17 2.9 Immunofluorescence (IF) staining 19 2.10 ROS assay 20 2.11 Image quantization 21 2.11.1 ROS quantification 21 2.11.2 Protein quantification 21 2.11.3 Statistical analysis 21 Chapter 3 Results and Discussion 23 3.1 Human LF cells express fibroblast-related proteins 23 3.2 The expression of TGF-β1 and TSP-1 in LF tissue 24 3.3 Mechanism of LSKL and alleviation of LF fibrosis 28 3.4 FG-4592 effect ROS and TSP-1 expression in LF cells 30 3.5 Effects between TSP-1 and ROS in LF cells 32 3.6 Interaction between TSP-1 and latent TGF-β1 36 Chapter 4 Discussion 39 Chapter 5 Conclusions 41 Chapter 6 Future works 43 References 44

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