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研究生:蕭惠禎
研究生(外文):Hui-Chen Hsiao
論文名稱:利用mPEG-PSLA 溫度敏感性水膠培養關節軟骨細胞層片
論文名稱(外文):Culture Chondrocytes Cell Sheet on mPEG-PSLA Thermosensitive Hydrogel
指導教授:魏毓宏朱一民朱一民引用關係
指導教授(外文):Yu-Hong WeiI-Ming Chu
口試委員:劉繼賢陳博彥
口試委員(外文):Chi-Hsien LiuBor-Yann Chen
口試日期:2014-07-17
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:87
中文關鍵詞:細胞層片溫度敏感性水膠關節軟骨細胞
外文關鍵詞:Cell sheetThermo-sensitive hydrogelChondrocytes cell
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  現代再生醫學大多是將細胞懸浮液注射進需要修補的組織或器官當中,或是另外用一種生物可降解的支架,將細胞培養於此支架內,直接將其移植到所需的部位當中但是懸浮液中的細胞易隨著體液流動而無法在固定處進行增生,進而醫治缺損部位,而支架又易使人體產生排斥,造成發炎反應,這使再生醫學上面臨極大的障礙,而細胞層片技術的利用,能避免上述的弊端,而且又能加速受損處的癒合,而溫度敏感性的培養品能避免利用胰蛋白酶切除細胞層片時對細胞外間質所造成的破壞,而細胞外間質的完整能加速細胞層片黏合於傷口上,並縮短其復原的時間。

  本實驗利用mPEG-PSLA溫度敏感性水膠培養關節軟骨細胞,此種水膠在5%時即可成膠,這可降低細胞的死亡率,擴大細胞的生長範圍,但5%的水膠在37℃的穩定度不如預期,因此測驗了5%、10%、15%及20%四種不同的濃度。將軟骨細胞種植於水膠上後,利用水膠溫度敏感的特點降低環境的溫度至4℃,使水膠轉變為液體型態後,觀察是否能將細胞層片完整取下,進而貼附於受損部位使其進行修復。
Recently, tissue engineering has long been regarded to possess huge potential ; conventional applications using biodegradable scaffolds have limited the tissue engineering field’s progress. Demonstrating a need for new methods. With success in clinical trials, regenerative therapies using these cell sheet approaches have therefore attracted significant interest and attention. As a novel alternative, we developed cell sheet engineering using temperature-responsive culture dishes, which allow non-invasive cultured cells as intact sheets along with their deposited extracellular matrix. Using this approach, cell sheets can be not only directly transplanted to host tissues without the use of scaffolding or carrier materials, but also used to create in vitro tissue constructing via the layering of individual cell sheets. Hence, cell sheet engineering allows tissue regeneration by either direct transplantation of cell sheets to host tissues or the creation of three-dimensional structures via the layering of individual cell sheets. By avoiding any additional materials such as carrier substrates or scaffolds, the complications associated with traditional tissue engineering approaches such as host inflammatory responses to implanted polymer materials, can be avoided. Proteolytic enzymes can be avoided by testing mPEG-PSLA hydrogel was tested for harvesting chondrocytes cells after carrying out cell culture at 37 °C and incubating the confluent cells at 4°C for spontaneous detachment of cell sheet from hydrogel .
中文摘要…………………………………………………………………I
Abstract………………………………………………………………III
誌謝……………………………………………………………………V
目錄……………………………………………………………………VI
表目錄…………………………………………………………………IX
圖目錄…………………………………………………………………X
第一章、序論……………………………………………………………1
第二章、文獻回顧………………………………………………………3
一、 細胞層片(Cell Sheet)………………………………3
1. 細胞層片在組織工程的應用…………………………………6
2. 細胞層片技術的優缺點………………………………13
二、 軟骨 ( Cartilage )……………………………………16
1. 透明軟骨 ( Hyaline cartilage ) ………………………………16
2. 彈性軟骨 ( Elastic cartilage ) ………………………………17
3. 纖維軟骨 ( Fibrocartilage ) ………………………………18
三、 製備細胞層片用之材料……………………………22
1. 水膠 ( hydrogel ) ……………………………………………22
1.1 水膠簡介………………………………………………22
1.2 環境刺激敏感性水膠分類…………………………23
1.3 mPEG-PSLA ……………………………………………34
第三章、實驗材料與方法………………………………………………40
一、 實驗架構……………………………………………40
二、 實驗藥品……………………………………………41
三、 實驗儀器……………………………………………41
四、 實驗方法……………………………………………43
1. 細胞培養……………………………………43
2. mPEG-PSLA高分子………………………43
3. 臨界微胞濃度分析…………………………43
4. 體外降解實驗………………………………47
5. 水膠溶液相轉變行為………………………48
6. pH值………………………………………52
7. 細胞存活率…………………………………52
8. 細胞毒性測試………………………………53
第五章、結果與討論……………………………………………………56
一、 高分子合成之供聚物水膠…………………………56
1. 臨界微胞濃度(CMC)量測……………………56
2. 高分子體外降解…………………………………57
3. 水膠相轉變行為…………………………………58
4. pH值……………………………………………62
二、 培養軟骨細胞層片…………………………………64
1. Live/Dead…………………………………………64
2. MTT測試…………………………………………71
3. 細胞層片……………………………………………72
第六章、結論與未來展望………………………………………………76
參考文獻………………………………………………………………78
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