2016-01-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657451摘要：研究背景：黃韌帶退化會導致椎管狹窄症，常伴隨神經性跛行、下背疼痛與功能限制等症狀，是老年人常見的脊椎退化性疾病，我們已證明彈性纖維降解與力學刺激是造成黃韌帶退化的潛在機轉。糖尿病是慢性代謝異常疾病，體內長期血糖上升，會造成多重系統併發症，臨床研究證實糖尿病與黃韌帶退化有關，然而，糖尿病造成黃韌帶退化的病理機轉仍待進一步研究。高濃度葡萄糖與糖化終產物(advanced glycation end products, AGEs)對細胞具多面向效應，包括影響基質蛋白、基質金屬蛋白酶與生長因子合成，調控骨母細胞相關基因表現，以及導致細胞凋亡，回顧目前已發表的文獻，並無高濃度葡萄糖與糖化終產物對黃韌帶細胞作用效應的相關探討，此實驗模式有助於了解糖尿病影響黃韌帶退化的分子機轉。研究目的：探討糖尿病影響黃韌帶退化的分子機轉，應用體外細胞培養模式，探討高濃度葡萄糖與糖化終產物對黃韌帶細胞代謝、骨化與凋亡的效應。本計劃預計將分三階段完成，第一年計劃為『探討高濃度葡萄糖與糖化終產物對黃韌帶細胞代謝的效應』；第二年計劃為『探討高濃度葡萄糖、糖化終產物與彈性蛋白對黃韌帶細胞骨性分化的效應』；第三年計劃為『探討高濃度葡萄糖、糖化終產物與力學刺激對黃韌帶細胞凋亡的效應』。研究方法：本計畫取得人體試驗委員會的執行許可後，取脊椎減壓手術的黃韌帶組織進行後續研究。第一年以組織切片分析AGEs 與AGE 接受體在黃韌帶組織的表現情形；以細胞培養模式，探討不同濃度葡萄糖與AGEs 對糖尿病與非糖尿病黃韌帶細胞代謝的影響，分析細胞活性、細胞毒性、自由基與特定基因表現。第二年以高濃度葡萄糖、AGEs 與彈性蛋白共同作用誘導黃韌帶細胞骨化，分析骨母細胞相關基因表現、骨性分化標記ALP 與Alizarin Red-S、酵素免疫反應測試以及訊息傳遞路徑MAPKs 與NF-kB。第三年以高濃度葡萄糖、AGEs 與力學刺激共同作用探討對黃韌帶細胞凋亡的影響，分析細胞凋亡外部與內部路徑caspase-3、6、8 和9，細胞凋亡相關基因TNFα、TNFR2、Fas、FasL 與抗氧化基因SOD 與GSH-PX 表現、自由基ROS 與NO 測定、訊息傳遞路徑MAPKs 與粒腺體蛋白Bax、Bcl-2 與Bid 分析。<br> Abstract: Background: Degeneration of ligamentum flavum contributes to the development of spinal stenosiswhich is a progressive degenerative disorder in elderly patients and frequently causes neurogenicclaudication, low back pain and functional limitation, etc. It was found in our previous study that elastindegradation products and mechanical stress are plausible mechanisms involved in the degeneration ofligamentum flavum. Diabetes mellitus (DM) is a chronic metabolic disease characterized by persistenthyperglycemia that has multi-system sequelae. Clinical studies have shown the associations betweenDM and the degeneration of ligamentum flavum. However, the pathomechanism of ligamentum flavumdegeneration caused by DM remained to be investigated. High glucose concentration and advancedglycation end products (AGEs) have multifaceted effects on cells, including synthesis of matrix protein,matrix metalloproteinase and growth factor, regulation of osteoblast-related gene expression, as well asleading to apoptosis. To our knowledge, there has been no report attempting to investigate the effects ofhigh glucose concentration and AGEs on ligamentum flavum cells in vitro. This experimental modelhelps to understand the molecular mechanisms by which DM affects the degeneration of ligamentumflavum.Purpose: The study aims to elucidate the molecular mechanisms by which DM affects the degenerationof ligamentum flavum and to further investigate the effects of high glucose concentration and AGEs onligamentum flavum cell metabolism, osteogenesis and apoptosis in vitro. The study proposed will beconducted in three consequent stages through a three-year period. In the first year, we plan toinvestigate the effects of high glucose concentration and AGEs on ligamentum flavum cell metabolism.In the second year, we plan to investigate the effects of high glucose concentration, AGEs andelastin-derived peptides on ligamentum flavum cell osteogenesis. In the third year, we plan toinvestigate the effects of high glucose concentration, AGEs and mechanical stress on ligamentumflavum cell apoptosis.Methods: After receiving approval of the Ethics Committee, ligamentum flavum specimens werecollected from patients undergoing spinal decompression surgery. In the first year, the expression ofAGEs and AGE receptor between diabetic and non-diabetic tissue samples were assayed by histologicalanalysis. Different concentrations of glucose and AGEs in culture medium were administered tomonolayer culture of diabetic and non-diabetic ligamentum flavum cells. The viability, cytotoxicity,free radicals and specific gene expression were evaluated. In the second year, high glucoseconcentration, AGEs and elastin-derived peptides were administered to monolayer culture of humanligamentum flavum cells. Gene expression of osteogeneic markers, osteogenic phenotypes, such asalkaline phosphatase and Alizarin Red-S stain, ELISA, as well as MAPKs and NF-kB signalingpathways were evaluated. In the third year, high glucose concentration, AGEs and mechanical stresswere administered to monolayer culture of human ligamentum flavum cells. Intrinsic and extrinsicapoptotic pathways were analyzed through caspase-3, 6, 8 and 9 activation. Gene expression of cellapoptosis, TNFα, TNFR2, Fas and FasL, and antioxidative enzymes, SOD and GSH-PX, were analyzedby RT-PCR. Reactive oxygen species, nitric oxide, signaling pathways of MAPKs as well asmitochondrial control of apoptosis, Bax, Bcl-2 and Bid, were further evaluated.糖尿病黃韌帶退化代謝骨化凋亡Diabetes mellitusligamentum flavum degenerationmetabolismosteogenesisapoptosis