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研究生: 梁晏銘
Yan-ming Liang
論文名稱: 高壓氧和高壓空氣對軟骨組織工程的影響
The effect of hyperbaric oxygen and air in cartilage tissue engineering
指導教授: 洪伯達
Po-Da Hong
戴念梓
Niann-Tzyy Dai
口試委員: 高震宇
none
學位類別: 碩士
Master
系所名稱: 應用科技學院 - 醫學工程研究所
Graduate Institute of Biomedical Engineering
論文出版年: 2011
畢業學年度: 99
語文別: 中文
論文頁數: 52
中文關鍵詞: 高壓氧高壓空氣軟骨組織工程
外文關鍵詞: hyperbaric oxygen, hyperbaric air, cartilage tissue engineering
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由於關節軟骨組織修復能力有限,對於罹患關節疾病的病人來言,發展組織工程軟骨是有其必要性。本研究使用脂肪來源之人類成體幹細胞,配合軟骨誘導培養液及明膠(gelatin)/聚己內酯(polycaprolactone)材料,用以探討高壓氣體環境對其組織工程軟骨之影響。實驗結果顯示,雖然各氣體壓力環境刺激的細胞生長速度低於控制組,但高壓氧及高壓空氣對GAGs濃度的濃度提升有明顯幫助。而透過Real-time PCR 分析結果發現2.5ATA air及2 ATA oxygen 環境刺激能夠加速提升軟骨特徵基因-SOX9、aggrecan、collagen II。綜合細胞生長、基質分泌及軟骨基因之表現,在2.5 ATA air 和2 ATA oxygen環境刺激下能幫助軟骨細胞生成及基質合成,具有應用於軟骨缺陷修復之潛力。


Owing to of the limited repair capacity of articular cartilage, it is essential to develop tissue-engineered cartilage for patients suffering from joint disease. Human adipose tissue derived stromal cells (ASCs) were seeded on the gelatin/polycaprolactone biocomposites to study the effects of hyperbaric oxygen and air on cartilage tissue engineering.
The results show the chondrocytes under hyperbaric oxygen and air’s stimulation, in histological analyzes, cartilage matrix was observed in groups and increased over 15 days; glycosaminoglycans syntheses have significant increase comparing to control;chondrogenic-specific gene expression of type II, aggrecan, and SOX9 was increased in until the fifteenth day.
Based on the data of cell proliferation, matrix accumulation and gene expression, 2.5 ATA air group and 2 ATA oxygen group contribute to chondrogenesis and cartilage matrix syntheses, both groups have potential in cartilage regeneration.

目 錄 中文摘要………………………………………………..………Ⅰ 英文摘要………………………………………….…………..Ⅱ 致謝…………………………………………………….…Ⅲ 目錄…………………………………………………………Ⅳ 圖表目錄……………………………………………………Ⅷ 第一章 前言…... .…………………………… …………….…………...1 1.1 關節軟骨疾病…………………………………..………....…... …..1 1.2 關節軟骨修復技術……………………..………. ….………… .. ...1 1.3 軟骨組織工程……………………..………. ……….….……….. ...2 1.4 軟骨組織……………………..………. ………….…….…. …… ...3 1.4.1軟骨基質………………..……. …………. ………….…….… …4 1.4.2 軟骨細胞………………..………. ………. ……..….…….…. …8 1.5 幹細胞在軟骨組織工程之應用………………..………. ……... …9 1.5.1 幹細胞來源與分類 ………………..………. …….…….…. …9 1.5.2 脂肪幹細胞………………..………. ………….……. ….…. …10 1.6 生醫材料在軟骨組織工程應用……………..……….…….…. …10 1.6.1 明膠簡介………………..………. ………..……….…….…. …12 1.6.2 明膠製作………………..………. ………..……….…….…. …13 1.6.3 聚己內酯簡介………………..………. ………….…. ….…. …13 1.7 訊息因子在軟骨組織工程之應用………. ………….…. ….…. ..14 1.7.1 高壓氧簡介……………. …. …………. ………….…. ….…. ..15 第二章 研究目的與實驗設計………. …. …………. …………. …. ..16 2.1 研究目的………………..………. ………..……….……….…. …16 2.2 實驗設計………………..………. ………..……….……….…. …16 第三章 材料與實驗方法………. …. …………. ………………. …. ..19 3.1 明膠與聚己內酯生物性複合材料的製備方法……………. …. ..19 3.2 人體脂肪幹細胞培養………. …. …………. ……..………. …. ..19 3.3 人類脂肪幹細胞鑑定………. …. …………. …………..…. …. ..19 3.4 脂肪幹細胞在生物相容材料上的培養及環境調控…..…. …. ....20 3.5 生物性質分析………. …. …………. …………..…………. …. ..21 3.5.1 細胞增生(Cell proliferation)…….….…………..…... …. ..21 3.5.2 阿爾新藍染色(Alcian blue stain) …………....…………….….22 3.5.3 GAGs分析-DMMB assay…………....……………….…...…..23 3.5.4 RNA 抽取與cDNA 合成………....……………………….…..23 3.5.5 及時定量PCR (Quantifcation Real-Time PCR,Q-PCR) .. ...24 3.6動物實驗……. …. …………. …………..…………..………. …. ..25 3.6.1動物條件及來源 (Animal Condition) ……..………. …. …...25 3.6.2 材料準備及動物手術模式 (Animal Model) .………. ….…....26 3.7 統計分析……. …. …………. …………..…………..………. …. 26 第四章實驗結果…. …. …………. …………..…………..………. …. 27 4.1脂肪幹細胞鑑定…………………………………………..…….….27 4.2 生物性質分析…………. ….………...…………..…………. …. ..27 4.2.1 細胞增生(Cell proliferation)…….….…………..…..... …. ..27 4.2.2 阿爾新藍染色(Alcian blue stain) …………....…………….….27 4.2.3 GAGs分析-DMMB assay…………....……………….…...…..28 4.2.4 及時定量PCR (Quantifcation Real-Time PCR,Q-PCR) .. ...28 4.3動物實驗……. …. …………. …………..…………..………. …. ..30 第五章結果討論…. …. …………. …………..…………..………. …. 27 5.1脂肪幹細胞鑑定…………………………………………..……….31 5.2 生物性質分析…………. ….………...…………..…………. …. ..31 5.2.1 細胞增生(Cell proliferation)…….….…………..….... ….. ..31 5.2.2 阿爾新藍染色(Alcian blue stain) …………....…………….….32 5.2.3 GAGs分析-DMMB assay…………....……………….…...…..32 5.2.4 及時定量PCR (Quantifcation Real-Time PCR,Q-PCR) .. ...33 5.3動物實驗……. …. …………. …………..…………..………. …. ..34 第六章結論與未來展望……………………..…………..………. …. 35 6.1結論…………………………………………………..……….35 6.2 未來展望..…………. ….………...…………..…………. …. ..35 第七章參考文獻………………. …………..…………..………. ……. 36

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