- 學位論文
搭載兒茶素之玻尿酸及膠原蛋白 複合物水膠於豬關節軟骨細胞模型 之抗發炎作用
Anti-inflammatory Effect of Catechin-Loaded Composite Hydrogel of Hyalurouic acid and Collagen on In-vitro Model of Porcine Articular Chondrocyte
摘要
摘要 本研究動機是利用綠茶萃取物兒茶素抑制軟骨細胞受到一氧化氮的氧化壓力；以膠原蛋白與甲基丙烯酸環氧丙酯改質玻尿酸的半互穿水膠搭載兒茶素，評估其對軟骨細胞的細胞毒性。軟骨細胞是萃取自豬大腿關節，以2.5 mg/ml 第二型膠原蛋白酵素在37 oC下使關節軟骨組織碎片的細胞游離，細胞經聚合酶鏈反應及電泳確認表達SOX-9、Aggrecan、type II collagen等軟骨分化特異性基因。一氧化氮為關節炎主要的發炎介質之一，研究中利用硝普鈉(Sodium nitroprusside，SNP)於培養液中釋放一氧化氮作為刺激因子，建立關節炎的體外細胞模型，搭配萃取自綠茶的兒茶素是否能保護細胞免於一氧化氮的損害。Griess assay測量結果顯示添加兒茶素的組別，一氧化氮的生成量較沒有添加兒茶素的組別降低50 %以上，可知兒茶素可抑制經硝普納釋放的一氧化氮。以瓊質擴散法，搭載兒茶素之複合薄膜與軟骨細胞共培養24小時，兒茶素搭載之複合薄膜具有良好的生物相容性。玻尿酸及膠原蛋白複合物經光交聯做為軟骨細胞的培養支架，本研究發現該薄膜可模擬軟骨細胞生長環境，再藉由兒茶素的抗氧化特性，降低一氧化氮的含量，而達到軟骨保護的效果。
並列摘要
Abstract Motivation for this study is the use of green tea extract EGCG inhibited chondrocytes by nitric oxide oxidative stress; To collagen and glycidyl methacrylate modified hyaluronic acid semi-interpenetrating hydrogel laded catechin, assess their impact on chondrocytes cytotoxicity. Cartilage cells are extracted from porcine articular to 2.5 mg / ml collagen type II enzyme in 37 oC under the articular cartilage cell debris free,cells were confirmed by polymerase chain reaction and electrophoresis express SOX-9、Aggrecan、type II collagen chondrocytes differentiation-specific genes. Nitric oxide is a major inflammatory mediators for osteoarthritis, studies using sodium nitroprusside (SNP) in the culture medium release of nitric oxide as a stimulating factor, with extracted from green tea catechins protect cells from nitric oxide damage.The adding catechin group NO volume will be 50 % lower than that not adding catechin group. Use agar diffusion method equipped with catechins composite film and chondrocyte were co-cultured for 24 hours, cells can be observed under the microscope adhesion and proliferation, the composite film is biocompatible. Equipped with catechins. hyaluronic acid and collagen by photo-crosslinking as culture scaffolds, this study found that the film can be simulated environment for the growth of chondrocyte, and then by catechin antioxidant properties reducing the levels of nitric oxide, suggesting a novel approach for chondroprotection.