林峰輝臺灣大學:醫學工程學研究所陳孟珮Chen, Moon-PeiMoon-PeiChen2010-06-022018-06-292010-06-022018-06-292008U0001-2906200912041400http://ntur.lib.ntu.edu.tw//handle/246246/184614退化性關節炎是一種和老化相關的疾病，它會造成患者的疼痛及行動不便。由於關節軟骨處於無血管、無神經的環境，並且代謝速率緩慢，因此軟骨的自我修復能力並不良好。抑制關節炎軟骨中不正常的細胞外基質降解，以及維持軟骨細胞的正常表現，是目前研究治療關節炎的一個方向。由基是一種高反應性的化學物質，在體內的形成和發炎免疫反應有關。雖然體內自由基的偵測並不十分容易，但在關節炎患者體內，可以檢查出一些過氧化的產物以及被硝基化的第二型膠原蛋白，顯示自由基對於關節炎的病理過程有著相當程度的影響。除此之外，越來越多研究證實，自由基影響了關節炎軟骨中不正常的代謝因子訊息傳遞。由於研究指出，在退化性關節炎者的體內所偵測到清除雙氧水的酵素，含量較正常者為低，因此雙氧水的累積是可以預期的。本篇研究的第一部分，即以雙氧水作為自由基的模型，測試雙氧水對於軟骨細胞基因表現的調控。結果顯示，被雙氧水所刺激的軟骨細胞，其發炎前驅因子 IL-1、TNF-α，以及金屬蛋白酵素 MMP-1、MMP-13的基因表現皆被向上調控。另一方面，維持軟骨細胞顯性的SOX-9基因則是被向下調控。這樣的基因表現與退化性關節炎軟骨細胞的表現，有著極為類似的趨勢。這些數據將雙氧水的影響與退化性關節炎的形成，做了一個更直接的連結。研究的第二部分，則是測試中藥抗氧化劑阿魏酸，對於被雙氧水調控的細胞是否有保護以及治療的能力。阿魏酸的抗氧化能力被證實比維生素B，維生素C，以及胡蘿蔔素還優異。經由WST-1, LDH, 以及 crystal violet的細胞活性測試，初步的實驗數據顯示阿魏酸對於軟骨細胞並沒有明顯的細胞毒性。在基因表現的調控上，預先加入阿魏酸以及事後加入阿魏酸，對於被雙氧水向上調控的發炎前驅因子IL-1、TNF-α，以及金屬蛋白酵素 MMP-1、MMP-13，皆有抑制的效果。而在ＳＯＸ－９的表現上面則是有部分回復的作用。研究的第三部分，是將阿魏酸更進一步的混合在一般用來輔助治療關節炎的透明質酸中，評估添加阿魏酸是否可提升透明質酸對於軟骨保護以及治療的效果。結果顯示，和無添加物的透明質酸相比，添加阿魏酸的透明質酸，對於被雙氧水刺激的發炎前驅因子IL-1、TNF-α，以及金屬蛋白酵素 MMP-1、MMP-13皆有向下調控的效果；並且對ＳＯＸ－９的基因表現亦可以向上調控。上的數據結果證實，雙氧水的確可能參與了退化性關節炎軟骨細胞的病理形成；而阿魏酸對於被雙氧水刺激的軟骨細胞，有保護以及治療的功能。更甚者，添加阿魏酸的透明質酸，讓透明質酸除了單純提供關節潤滑的同時，亦有著保護軟骨細胞不受自由基影響，以及將受雙氧水刺激的細胞調整回正常的潛力。Osteoarthritis is one of the major degenerative diseases which affects millions people these days. Due to the tissue specificity, such as low turnover rate and avascular environment, the self-repair of cartilage is considered to be limited. For the intervention of cartilage destruction in OA, inhibition of enzymatic degradation of extracellular matrix (ECM) as well as maintaining the cellular phenotype is one of the major goals of interest. ree radicals, including reactive oxygen speicies (ROS) and reactive nitrogen species (RNS), are the active biochemical compounds that associated with the chronic inflammatory reaction in osteoarthritis. Observation of lipid peroxidation products and nitrated collagen peptide in the serum of patient implicates the role of free radicals in the pathology osteoarthritis. he first part of this study is to use to hydrogen peroxide to provide the oxidative stress and assess the gene expression of chondrocytes cultured within it. Results from real-time PCR reveal that incubated chondrocytes in hydrogen peroxide in either short term or long term did up-regulate the inflammatory gene IL-1 and TNF-α expression in mRNA level. It also triggered the expression of metalloproteinase MMP-1 and MMP-13, which are both crucial enzymes responsible for cartilage destruction in OA. The down-regulation of SOX-9 also implicates the involvement of ROS in the loss of phenotype in osteoarthritic cartilage. The similar gene expression pattern between hydrogen peroxide-stimulated chondrocytes and osteoarthritic chodrocytes provides stronger evidence that link ROS to the pathology of OA. he second part in my study is to use ferulic acid, the natural antioxidant compound from Chinese herb Angelica (Dong-Gui) which is reported to have excellent antioxidant property compared to γ-tocopherol, α-carotene, and ascorbic acid, to retrieve the effects result from excess produced free radical. In our preliminary results, ferulic acid shows low cytotoxicity on chondrocytes according to the WST-1, LDH, and crystal violet assay. Within the safety concentration, pre-treat ferulic acid can down-regulate hydrogen peroxide-induced IL-1, TNF-α, MMP-1 and partially restore SOX-9 gene expression in mRNA level. Post-treatment of ferulic acid also intervened MMP-1, MMP-13 gene expression showing the potential of ferulic acid blocking the unusual catabolic activity that trigger by hydrogen peroxide.urthermore, while HA alone did not do much effect on hydrogen peroxide-treated chondrocytes, ferulic acid containing HA gel showed protective and therapeutic effects by down-regulating IL-1, TNF-α, MMP-1, MMP-13 and up-regulating SOX-9 gene expression in pre-treatment and post-treatment model.The result in this study provided stronger evidence that implicate hydrogen peroxide in the pathology of OA and shows the potential of ferulic acid- containing HA gel in providing extra protective and therapeutic effect in treatment of OA.TABLE OF CONTENTS謝……………………………………………………………………………………ii文摘要……………………………………………………………………………...iiibstract………………………………………………………………………………vable of content……………………………………………………………………viiiist of figures………………………………………………………………………xiiiist of tables………………………………………………………………………xvihapter 1 Introduction………………………………………………………………1.1 Preface: The degenerative disease: osteoarthritis………………………………1.2 Therapies for osteoarthritis……………………………………………………2.2.1 Non-pharmaceutical intervention……………………………………2.2.2 Pharmaceutical intervention…………………………………………2.2.3 Surgical options………………………………………………………3.3 Tissue engineering and osteoarthritis…………………………………………4.4 Purpose of study………………………………………………………………5hapter 2 Theoretical Basis…………………………………………………………6.1 Cartilage in human body………………………………………………………6 2.1.1 The adult articular cartilage: chondrocytes and extracellular matrix………………………………………………………………………6.1.2 The biology of chondrocytes…………………………………………8 2.1.3 The extracellular matrix: collagen, proteoglycans, and non-collagenous proteins………………………………………………………………………8.2 The anabolic and catabolic activities in chondrocytes…………………12 2.2.1 Inflammtory gene expression in osteoarthritis……………………14 2.2.2 Proteinases in cartilage degradation…………………………………14.3 Signal Transduction in Cartilage Homeostasis……………………………172.3.1 Signal pathway in chondrogenesis and the chondrogenic phenotype………………………………………………………………………………………………………………………………17.4 Osteoarthritis and Inflammation: Pro-Inflammatory Gene Expression and the involvement of inflammatory mediators………………………………………21.4.1 The Hypertrophic Phenotype in Osteoarthritic Chondrocytes………21.4.2 Proinflammatory cytokine and inflammatory mediators in OA………21.4.3 Signal Transduction in Response to Cytokines and Soluble Mediators of Inflammation: MAPKs………………………………………………………24.5 Free radicals and osteoarthritis……………………………………………26 2.5.1 The formation of free radicals………………………………………26 2.5.2 The natural antioxidant system ………………………………………28 2.5.2.1Enzymatic defense…………………………………28 2.5.2.2Non enzymatic defense……………………………30.5.3 Oxidative stress………………………………………………………32 2.5.4 Effect of oxidative stress on chondrocyte……………………………32 2.5.5 Free radicals in signal transduction……………………………………33 2.5.6 The redox-sensitive transcriptional factors……………………………33.5.7 Antioxidant therapy for osteoarthritis…………………………………35.6 Antioxidant in Chinese herb: ferulic acid ……………………………………42hapter 3 Material and method……………………………………………………44.1 Experimental set up……………………………………………………………44.2 Primary culture of chondrocyte from bovine articular cartilage……………51.3 The Evaluation of Cytotoxicity: Determination of Safety Concentration……52.3.1 Lactate Dehydrogenase (LDH) Assay………………………………52.3.2 WST-1 assay…………………………………………………………53.3.3 Crystal Violet Assay…………………………………………………54.4 Gene Expression Evaluation: Reverse- transcription PCR and Real-time PCR………………………………………………………………………………55.4.1 Extraction of RNA from Porcine Chondrocytes……………………56.4.2 Primer Selection for Target Gene by Gel Electrophoresis…………58.4.3 Gene Expression: Evaluation by Real-time PCR……………………58.5 Free Radical Detection…………………………………………………………59.5.1 Detection of Hydrogen Peroxide and Hydroxyl Radicals by Chemical lumen : Determination of Effective Concentration………………………61.4 UV Spectrometer: The Detection of Ferulic Acid Released from Hyaluronic Acid……………………………………………………………………………63hapter 4 Results and discussion……………………………………………………64.1 The examination of the selected primer: gel electrophoresis………………64.2 The effect of hydrogen peroxide on porcine chondrocytes in mRNA level………………………………………………………………67.3 Cytotoxicity of Ferulic Acid on Chondrocytes: The Evaluation of Safety Concentration………………………………………………………………72.4 Free Radical Detection: Determination of Effective concentration………75.5 The effect of ferulic acid on hydrogen peroxide- stimulated porcine chondrocytes in mRNA level: Short term evaluation…………………………76.6 The Release of Ferulic Acid from Ferulic Acid Containing Hyaluronic Acid Gel: Detection by UV Spectrometer…………………………………………80.7 The effect of ferulic acid containing HA gel on hydrogen peroxide- stimulated chondrocytes: long term evaluation……………………………76.7.1 The gene expression of chondrocytes when expose to a continuous oxidative stress environment ……………………………………………76.7.2 The effect of ferulic acid containing HA gel on hydrogen peroxide-stimulated chondrocytes: pre-treat in long term model…………79.7.3 The effect of ferulic acid containing HA gel on hydrogen peroxide-stimulated chondrocytes: post-treat in long term model………83hapter 5 Conclusion………………………………………………………………87eference……………………………………………………………………………89application/pdf1185146 bytesapplication/pdfen-US退化性關節炎自由基雙氧水阿魏酸抗氧化劑透明質酸發炎前驅因子osteoarthritisfree radicalhydrogen peroxidehyaluronic acidferulic acidantioxidantpro-inflammatroy cytokinesthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/184614/1/ntu-97-R93548029-1.pdf