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研究生:古莞霖
研究生(外文):Ku, Kian-Lin
論文名稱:以光交聯PEG/PCL水膠包埋法共同培養間葉幹細胞與軟骨細胞應用於軟骨組織修復
論文名稱(外文):Co-culture of Encapsulated Mesenchymal Stem Cells and Chondrocytes in PEG/PCL Photocrosslinked Hydrogel Scaffolds for Cartilage Tissue Engineering
指導教授:朱一民朱一民引用關係
指導教授(外文):Chu, I-Ming
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:115
中文關鍵詞:光膠聯水膠共培養軟骨細胞間葉幹細胞
外文關鍵詞:HydrogelCo-cultureChondrocyteMesenchymal stem cell
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隨著老年人及肥胖人口增加,退化性關節炎的病患也隨之增加。關節軟骨組織缺乏血管及淋巴管,其自我修復能力不如一般組織。依照組織工程的概念,將細胞取出後植入適當的支架培養,使其生成組織替代物後修補體內原本受損的部位,為未來關節軟骨疾病的重要療法。近年來,水膠已成為研究軟骨組織修復的支架型態之一,其高含水率有助於物質的代謝且提供適當的彈性可於修復初期支撐細胞。軟骨組織工程之細胞來源則可分為軟骨細胞及幹細胞。軟骨細胞除來源有限外,於體外培養容易去分化,使其分泌的細胞外基質近似纖維軟骨;而幹細胞單獨培養的誘導效率低且有腫瘤化的潛在風險,造成臨床使用上的限制。故本研究嘗試以共同培養此兩種細胞,以軟骨細胞誘導間葉幹細胞成軟骨細胞,提升透明軟骨細胞外基質的合成效率。
實驗將以PEG/PCL三團共聚物做為材料。將不同長度疏水鏈 (PCL)接枝於親水材料 (PEG)兩端,混合細胞後以UV光交聯成水膠,以紐西蘭兔之軟骨細胞與間葉幹細胞為細胞來源,分析水膠之物質且比較細胞包埋於不同親疏水比例之材料差異,分析其細胞外基質的合成(GAG及膠原蛋白)效率。選出較佳的親疏水比例材料後,進行軟骨細胞與間葉幹細胞共同培養。結果顯示細胞外基質的分泌會隨著時間增加,且依不同親疏水材料其合成效率也不同,其中以PECL12水膠材料較優異。由基因表現與組織染色之結果,發現以幹細胞與軟骨細胞比例為4:1 (S4C1)的比例共培養表現較佳。進一步以動物實驗八週後也可以發現,以PECL12水膠包埋S4C1比例的修復效果較佳。

The U.S. costs 128 million per year due to arthritis disease currently. With the aging and obesity, osteoarthritis patients have increased. Cartilage lacks blood vessels and lymphatic tissue, its ability to repair itself doesn’t like other tissue. According to the concept of tissue engineering, the cells were transplanted to the suitable scaffolds, to replace the damaged parts of the original by regenerative tissue. In recent year, hydrogels have been used as scaffolds for cartilage tissue regeneration, partly for their high water content that can enhance nutrients transport. Cartilage tissue regeneration was confronted with limited availability of suitable autologous chondrocytes, since in vitro expansion in 2-D culture of these cells leads to de-differentiation, and the loss of their ability to secrete cartilage extracellular matrices such as collagen type II and proteoglycan. The cell source of cartilage tissue engineering is chondrocytes and Mesenchymal stem cells (MSCs). MSCs cultured alone have low induced efficiency and potential risk of tumor formation, resulting in restriction on clinical use. This study is trying to induce MSCs chondrogenicly by co-culture with chondrocytes, which stimulate MSCs differentiation.
On the scaffold aspect, this study synthesized different molar ratio of PEG/PCL triblock copolymer, using different lengths of hydrophobic chains (PCL) block with the hydrophilic material (PEG) at both end. Cell compatibility of different hydrophobic/ hydrophilic ratio hydrogels was examined by analyzing the synthesis of extracellular matrix (such as GAG and collagen). The results showed that the secretion of extracellular matrix increase with time and have different synthesis ability with different hydrogels. The suitable materials were selected to culture with different ratio of chondrocytes and MSCs. These results suggested that the PECL12 hydrogels are suitable to encapsulate MSCs and chondrocytes. During co-culture experiment, different MSCs and chondrocytes ratio resulted in different ECM accumulation after 4 week. Compared with gene expression and histological analysis can found that S4C1 has better results than other co-culture ratio. Furthermore, using PECL12 hydrogels encapsulated S4C1 can found better regeneration efficiency in vivo.

摘 要 II
Abstract IV
誌謝 V
目錄 VII
表目錄 X
圖目錄 XI
第一章 文獻回顧 1
1.1 關節軟骨組織 1
1.1.1 關節軟骨細胞 2
1.1.2 關節軟骨結構 2
1.1.3 關節軟骨基質 4
1.1.4 關節軟骨受損及修復現況 5
1.2 關節軟骨組織工程 12
1.2.1 細胞 12
1.2.2 生長因子 13
1.2.3 支架 14
1.2.4 軟骨組織工程研究現況 17
1.3 三維光交聯水膠 19
1.3.1 光交聯水膠成膠機制 19
1.3.2 常見光交聯水膠材料 20
1.3.3 光交聯水膠的特性 21
1.3.4 光交聯水膠相關研究 22
1.4 間葉幹細胞 26
1.4.1 間葉幹細胞的軟骨分化與基因表現 26
1.4.2 間葉幹細胞於軟骨組工程之應用 27
1.4.3 水膠包覆間葉幹細胞的相關研究 28
第二章 研究動機與目的 32
第三章  實驗架構、藥品與儀器 34
3.1 實驗架構 34
3.2 實驗藥品 36
3.3 實驗儀器 38
3.4 基因序列 39
第四章 實驗步驟與方法 40
4. 1 PCL-PEG-PCL 合成 40
4.1.1 PCL-PEG-PCL三團共聚物合成 40
4.1.2 PCL-PEG-PCL、PEG兩端烯基化反應 40
4. 2 鑑定材料性質方法 41
4. 3 水膠製備 41
4. 4 含水率 42
4. 5 機械性質測試 42
4. 6 降解性測試 42
4. 7 細胞純化及培養 43
4. 8 細胞包埋 44
4. 9 細胞存活率測試 45
4. 10 Live/Dead 螢光染色 45
4. 11  生物性質分析 46
4.11.1 樣品前處理 46
4.11.2 DNA定量 46
4.11.3 GAG定量 47
4.11.4 Total collagen 定量 47
4.12 RNA 萃取與定量 48
4.13 RNA反轉錄cDNA 49
4.14 即時定量PCR 50
4.15 組織切片染色 50
4.15.1 脫臘處理 51
4.15.2 Hemotoxylin-eosin stain, H&;E stain 51
4.15.3 Alcian blue 51
4.15.4 Safranin O 51
4.15.5 膠原蛋白二型組織免疫染色 52
4.15.6 膠原蛋白一型組織免疫染色 53
4.15.7 膠原蛋白十型組織免疫染色 53
4.16 動物體內實驗 54
第五章 實驗結果與討論 55
5.1 材料合成與鑑定 55
5.2 材料物理特性 59
5.3 材料降解 59
5.4 細胞存活率 60
5.5 以水膠單獨培養軟骨細胞與間葉幹細胞 61
5.5.1 生物化學分析 62
5.5.2 基因表現 65
5.5.3 組織切片染色 67
5.5.4 綜合比較 82
5.6 以水膠共培養軟細胞與間葉幹細胞 83
5.6.1 生物化學分析 83
5.6.2 基因表現 85
5.6.3 組織切片染色 86
5.6.4 綜合比較 98
5.7 高細胞密度培養 102
5.7.1 生物化學分析 102
5.7.2 基因表現 104
5.7.3 組織切片染色 105
5.8 動物實驗 107
第六章 結論 110
參考文獻 112
附錄 115

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