以表面貼附程度和細胞間的作用來控制骨髓間葉幹細胞往軟骨分化之影響

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姓名

林聖倫(Sheng-lun Lin) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

以表面貼附程度和細胞間的作用來控制骨髓間葉幹細胞往軟骨分化之影響
(The Effect of Surface Adhesivity and Cell-Cell Contact on chondrogenesis of Mesenchymal Stem Cells)

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摘要(中)

水膠(Hydrogel)有很高的含水量和相對低的細胞貼附率。有許多研究指出水膠可提供微觀環境來做軟骨分化，特別的是有些還會將水膠混摻透明質酸和膠原蛋白。在我們的研究中，我們試著去調控表面貼附力和細胞間的接觸作用來誘導骨髓間葉幹細胞(RM1)往軟骨分化。
幾丁聚醣(Chitosan, CS)被選來適合細胞貼附的基底材料，另外我們使用表面改質，將兩端帶有Carboxylate的PEG(CP)接枝到CS表面上，以達到細胞貼附的改變。而在三維培養中，先製備好在不同孔徑大小的尼龍(Nylon mesh membrane)披覆上CS或是CP接枝上CS表面，來營造出一個三維的環境，利用此環境來控制細胞間的接觸作用，尼龍的孔徑大小我們選用30μm，100μm和180μm。在180μm的尼龍擁有較低的表面對體積的比率。在100μm和180μm中，我們觀察到細胞會逐漸聚集。在披覆CS的尼龍培養在Basal medium中，藉由GAGs染色和基因表現的分析指出有軟骨分化的趨勢。並且發現在180μm中對於間葉幹細胞的軟骨分化最好，也告知我們細胞間接觸作用的重要性。有趣的是，在披覆CS的尼龍培養在Chondrogenic medium中，並未大幅度地提升軟骨分化。
在CP的尼龍中，細胞在第六個小時就開始出現聚集的現象，而且Aggrecan和Col2a1有更高的表現量，若再添加Chondrogenic medium來培養，發現又更能促進RM1分化成軟骨細胞，GAGs染色和基因表現量都是最好的。
從這些結果指出當我們稍微的降低表面貼附率就可以增加細胞間的接觸作用，而且推論出促進軟骨分化的重要因素就是要提升細胞間的接觸作用。

摘要(英)

Hydrogels display high water retention and relatively low cell adhesivity. Several studies have demonstrated that hydrogels can provide an instructive microenvironment for the chondrogenesis of mesenchymal stem cells (MSC), especially when hyaluronan and collagen II were incorporated. In this study we try to elucidate that it is surface adhesivity, cell-cell contact, or bio-specific matrix-cell interaction dominates instructive differentiation.
Chitosan was selected as the base substrate for cell attachment. Cell adhesion was altered through surface modification of chitosan by carboxylate ended polyethylene glycol (CP). Nylon meshes of different mesh sizes coated with chitosan or CP modified chitosan were prepared to provide 3-D environments for cell-cell contact control. The size of Nylon mesh selected are 30, 100, 180μm. The 180μm mesh owns the lowest surface to volume ratio. We have observed that the cell would gradually aggregate in the wells of 100 and 180μm. Nylon mesh coated with chitosan even in basal medium. GAG staining as well as SOX9, Aggrecan, and Col2a1 gene expression indicate chondrogenesis. Results indicate that the 180μm mesh is the best for MSC chondrogenesis suggesting the importance of cell-cell contact. Interestingly, chondrogenic medium does not promote chondrogenesis when MSCs are cultivated in chitosan coated meshes.
In Nylon mesh coated with CP modified chitosan, we observe that cells aggregate within 6 hours in the wells of 100 and 180μm mesh. Much higher SOX9, Aggrecan, and Col2a1 expression are observed when MSCs are cultivated in Nylon mesh coated with CP modified than with virgin chitosan.
From these results, we find that slightly reduction of surface adhesivity enhances cell-cell contact. And the enhancement of cell-cell contact is critical to chondrogenesis.

關鍵字(中)

★ 聚乙醇
★ 骨髓間葉幹細胞
★ 軟骨分化
★ 幾丁聚醣

關鍵字(英)

★ MSCs
★ chondrogenesis
★ chitosan
★ PEG

論文目次

摘要 I
Abstract III
誌謝 IV
目錄 V
圖目錄 IX
表目錄 XIII
第一章緒論 1
1-1 研究動機 1
1-2 研究目的 2
第二章文獻回顧 3
2-1 軟骨 3
2-1-1 關節軟骨 4
2-1-2 組織工程應用於關節軟骨修復 7
2-2 幹細胞 9
2-2-1 間葉幹細胞 10
2-2-2 間葉幹細胞在組織修復上的應用 11
2-3 軟骨分化的特性 12
2-3-1 軟骨分化的生理機能 12
2-3-2 細胞與細胞之間與細胞與基材的作用 13
2-3-3 軟骨分化的基因表現 15
2-4 誘導幹細胞分化成軟骨細胞之體外方法 16
2-4-1 天然高分子材料 16
2-4-2 影響軟骨化的表面改質 18
2-4-3 生長因子對於軟骨影響 20
第三章實驗藥品、儀器與方法 22
3-1 實驗藥品 22
3-1-1 表面改質 22
3-1-2 細胞培養 22
3-1-3 固定細胞及染色 23
3-1-4 基因表現分析 23
3-2 實驗儀器 23
3-3 溶液配製 24
3-4 實驗方法 26
3-4-1 實驗架構 26
3-4-2 表面改質 27
3-4-2-1 幾丁聚醣薄膜的製備 27
3-4-2-2 羧基-聚乙二醇-羧基的接枝 29
3-4-3 細胞培養 29
3-4-3-1 繼代培養 29
3-4-3-2 凍存細胞 30
3-4-3-3 解凍細胞 31
3-4-4 細胞分化 31
3-4-4-1 二維培養 31
3-4-4-2 三維培養 32
3-4-5 Safranin O 染色 32
3-4-6 基因表現分析 33
3-4-6-1 萃取RNA 33
3-4-6-2 反轉錄(Reverse transcription) 33
3-4-6-3 定量聚合酶鏈鎖反應(Q-PCR) 35
第四章結果與討論 37
4-1 二維培養 37
4-1-1 細胞型態 37
4-1-2 Safranin O 染色 47
4-1-3 基因表現分析 49
4-2 三維培養 52
4-2-1 細胞型態 52
4-2-2 Trypan blue 染色 61
4-2-3 Safranin O 染色 62
4-2-4 基因表現分析 66
第五章結論 71
第六章參考文獻 72

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指導教授

阮若屈(Ruoh-Chyu Ruaan)

審核日期

2012-8-6

推文