臺灣博碩士論文加值系統

近年來，在先進國家人口的老年化及意外事故的增加，因老化或關節軟骨受損(Articular cartilage damage)所造成的關節軟骨退化(Articular cartilage degeneration)或退化性關節炎(Osteoarthritis)，已成為先進國家所重視的議題，目前台灣之老人慢性疾病中，退化性關節炎高居第七位。關節軟骨缺乏血流供應及幹細胞，使軟骨組織的自體修復相較困難。目前治療退化性關節炎的方式，僅消極的以非類固醇(NSAIDs)止痛消炎、關節內注射玻尿酸或口服醣胺素製劑之輔助性治療之外，並無其他較有效治療藥物以抑制關節軟骨之退化。本研究之目標是希望利用組織工程(Tissue engineering)積極治療及重建關節軟骨缺損。人類間質幹細胞的軟骨新生的過程中，細胞外間質(Extracellular matrix)，特別是膠原蛋白(Collagen)的改變，其透過膠原蛋白接受體對人類間質幹細胞走向軟骨新生，扮演著相當重要的角色。Discoidin Domain Receptor(DDR)為較新發現的膠原蛋白接受體，可調控上皮細胞的增生、貼附與分化，但其在促進脂肪幹細胞之軟骨新生中扮演的角色尚未被釐清。本研究利用pellet culture及軟骨誘導培養基(Chondroinduction medium)來誘導人類脂肪幹細胞產生軟骨新生。並偵測軟骨新生的重要標記基因表現 (SOX-9、Type II collagen與 Aggrecan)，及利用組織學分析(Alcian blue stain) 及1, 9-dimethylmethylene blue (DMMB) Assay來測量sulfates glycosaminoglycans(sGAG)的含量以確定軟骨化。在實驗中確定Chondro-induction組比control組有較佳的軟骨化基因表現例如Chondro-induction組(Ch-I)的SOX-9、Type II collagen與Aggrecan都有比Control組有較高的基因表現及sGAG的生成。進一步的觀察DDR1的基因及蛋白質表現顯示其在Ch-I組之第2天有顯著增加；然而 DDR2基因表現與蛋白質表現在實驗組與對照組則無顯著差異。因此我們進一步的利用DDR1 short hairpin RNA (shRNA)來抑制人類脂肪幹細胞的DDR1表現，首先在抑制DDR1表現(DDR1-depleted)的人類脂肪幹細胞上發現，於軟骨誘導環境中培養時，DDR1-depleted人類脂肪幹細胞較Mock組的細胞生長具顯著性抑制。在於軟骨新生方面，抑制DDR1表現的人類脂肪幹細胞顯示有更好的軟骨新生指標基因表現(SOX-9、Type II collagen與 Aggrecan)以及細胞外基質的生成(Type II Collagen與Glycosaminoglycans)。根據上述實驗結果，推論DDR1在誘導人類脂肪幹細胞軟骨化的過程中可能扮演著負向調控的角色。

Recent years, it has already become a critical topic in the developed country that the articular cartilage traumas result in the cartilage degeneration or osteoarthritis, especially in the population of old age and contingency. In Taiwan, osteoarthritis has occupies the seventh place of chronic disease in old man. Articular cartilage lacks the stem cells and the blood flow supplied, which do increased difficulty in the cartilage self-repairing from the body. At present, it still lacks effective treatment or medicine to heal the articular cartilage disease. Our goal is to use tissue engineering for treating and regenerating the articular cartilage defect. In the process of Chondrogenesis, the collagens are critical for modulating the chondrogenic differentiation of human mesenchymal stem cells. Discoidin Domain Receptors (DDRs), collagen receptor, regulate several cell behaviors, including cells adhesion and cell differentiations. However, the roles of DDRs in chondrogenesis of mesenchymal stem cells remain undefined. Pellet cultures with chondro-induction medium were used for inducing chondrogenesis of hMSCs, Effect of DDRs on chondrogenesis of human adipose tissue derived mesenchymal stem cells (hADSCs) were confirmed by analyzing, the protein expression (DDRs) with Western blot, the chondrogenetic gene (SOX-9, Type II collagen & Aggrecan) expression with Real-time PCR, and sulfates glycosaminoglycans (sGAG) with Alcian blue stain and 1,9-dimethylmethylene blue assay. Chondro-induction (Ch-I) triggered increasing chondrogenetic gene and sGAG expression in hADSCs compare with control group. DDR1 gene expressions in Ch-I group shows significantly different from control group, and DDR1 protein expression was elevated after chondroinduction in early stage, while DDR2 gene expression and protein expression were not. We abrogated DDR1 gene expression with DDR1 short hairpin RNA (shRNA). The DDR1-depleted hADSCs was suppressed cell growth in Chondroinduction medium. The result not only showed that the expression of chondrogenic marker genes (SOX-9, Type II collagen & Aggrecan) in DDR1-depleted group were gradually increased compared to Mock group, but also sGAG synthesis was augmented in DDR1-depleted hADSCs. In conclusion, our findings show DDR1 play a negative control in the Chondrogenesis of hADSCs.

中文摘要P.2
英文摘要P.5
緒論P.8
研究目的及研究設計P.18
材料及方法P.20
結果P.33
討論P.48
參考文獻P.53

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