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研究生： 歐哲佑
Ou, Che-Yu
論文名稱： 細胞外基質影響軟骨膜前驅細胞之軟骨分化特性之研究
Effects of extracellular matrix components on the chondrogenesis of perichondrium progenitor cells
指導教授： 黃玲惠
Huang, Lynn L.H.
學位類別： 碩士
Master
系所名稱： 生物科學與科技學院 - 生物科技研究所
Institute of Biotechnology
論文出版年： 2010
畢業學年度： 98
語文別： 中文
論文頁數： 103
中文關鍵詞： 軟骨膜前驅細胞 、細胞外基質 、軟骨分化
外文關鍵詞： Perchondrium derived progenitor cells, extracellular matrix, chondrogenesis
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    • 針對軟骨缺陷的疾病，目前可藉由組織工程的方式來進行治療，不過需將幹細胞於體外誘導走向軟骨分化，等細胞數達到一定量後再進一步植回病患受損處。由於軟骨組織具有大量的天然細胞外基質，而這些基質可以提供幹細胞更接近天然軟骨的環境，然而目前尚無文獻針對諸多不同種類的基質，對於軟骨分化的影響進行完整的探討。相較於其他的幹細胞，軟骨膜前驅細胞較傾向走向軟骨分化，且較容易從外科手術中取得，本實驗室先前已建立分離軟骨膜前驅細胞(PCPC)的技術。因此本篇論文的實驗目標即為探討胞外基質對於軟骨膜前驅細胞之軟骨分化之影響。
    本篇論文的實驗規劃可分為三個階段，分別為 (1). 探討PCPC 的特性，從實驗結果中可以推測出，PCPC極有可能為處於細胞聚集 (cell condensation) 時期的幹細胞，且可進一步往下分化成軟骨細胞；(2). 快 速地篩選眾多的實驗組別，以尋找能夠促進PCPC走向軟骨分化的 最佳條件，從實驗結果中發現，將PCPC培養於serum-free DMEM-low glucose，且不論是單獨或是混合使用玻尿酸、明膠以及纖維黏蛋白之環境中七天，即會分化成軟骨細胞；以及 (3). 建立PCPC的無血清培養環境。綜合以上實驗結果，我們以建立PCPC之鑑定方法，且證實細胞外基質(如:玻尿酸、明膠與纖維黏蛋白)有助於PCPC的軟骨分化。

    The disease of cartilage defects can be treated by tissue engineering. However, stem cells need to be induced to differentiate into chondrocytes, and cell number has to reach certain amount before they can be transplanted onto injury site.
    In our lab, we establish the method of isolating perichondrium progenitor cells (PCPCs). In contrast to other stem cells, PCPCs prefer to proceed with chondrogenesis, and are technically easier to be isolated by operation. Furthermore, cartilage has a large amount of extracellular matrix, this extracellular matrix could support a suitable environment for stem cell differentiating into chondrocyte. However, research of ECM on chondrogenesis is still lacking, therefore, the aim of this paper is that extracellular matrix components effect on the chondrogenesis of PCPCs.
    In this paper, we design three parts of experiment. First, we want to know the characteristic of PCPC. From the results it can be predicted that PCPC may be the stem cells in the condensation phase. Second, we want to create the microenvironment, which is constituted of multi- matrices, and to study effects of microenvironment on the chondrogenesis of perichondrium progenitor cells. From the results, we know that HA, gelatin and fibronectin can induce PCPC differentiation into chondrocyte. Third, we want to create the serum-free culture system. Finally, we know the characteristic of PCPC, and add HA, gelatin and fibronectin which can promote chondrogenesis of PCPC. It also proves that ECM can promote chondrogenesis.

    目 錄 中文摘要 -3- 英文摘要 -4- 誌謝 -6- 符號及縮寫 -7- 目錄 -8- 表目錄 -12- 圖目錄 -13- 正文 : 一. 研究背景與目的 1.1 軟骨組織簡介 -15- 1.1.1 軟骨組織在人體的分佈及特性 -15- 1.1.2 軟骨組織在不同部位之細胞型態 -15- 1.1.3 軟骨膜之分化潛能 -16- 1.1.4 軟骨新生 -16- 1.1.4.1 軟骨新生簡介 -16- 1.1.4.2 細胞外基質對於軟骨新生影響 -17- 1.2 軟骨膜前驅細胞簡介 -18- 1.2.1 軟骨膜前驅細胞之定義 -18- 1.2.2 軟骨膜前驅細胞的鑑定及特性分析之相關文獻回顧 -19- 1.2.2.1 細胞及分子標誌 -19- 1.2.2.2 軟骨膜前驅細胞分化潛力之探討 -20- 1.3 軟骨相關疾病及目前治療方式 -21- 1.3.1 退化性關節炎 -21- 1.3.2 整形外科 -22- 1.3.3 幹細胞治療的優勢 -22- 1.3.4 軟骨膜前驅細胞治療新希望 -22- 1.4 胞外基質對於軟骨相關細胞之影響 -23- 1.4.1 天然胞外基質的重要性 -23- 1.4.2 誘導幹細胞分化成軟骨細胞之體外試驗方法 -23- 1.4.2.1 細胞激素及生長因子對於軟骨影響之文獻回顧 -24- 1.4.2.2 天然或合成之細胞外基質對於軟骨影響之文獻回顧 -24- 1.5 實驗目標 -25- 1.6 實驗設計 -25- 二. 研究的方法以及步驟 2.1 藥品及材料 -27- 2.1.1 軟骨膜相關細胞以及軟骨細胞萃取 -27- 2.1.2 細胞培養及冷凍 -27- 2.1.3 細胞外基質 -28- 2.1.4 染劑 -28- 2.1.5 免疫染色試劑及耗材 -29- 2.1.6 基因表現量測定 -29- 2.1.6.1 RNA 萃取 -30- 2.1.6.2 RT-PCR -30- 2.1.6.3 q-PCR -30- 2.2 儀器設備 -31- 2.3 方法 -32- 2.3.1 軟骨膜相關細胞以及軟骨細胞萃取 -32- 2.3.1.1 藥品配置 -32- 2.3.1.2 步驟 -33- 2.3.2 細胞外基質coating -37- 2.3.2.1 多醣體基質 -37- 2.3.2.2 蛋白質基質 -38- 2.3.3 染劑染色 -39- 2.3.3.1 藥品配置 -39- 2.3.3.2 步驟 -39- 2.3.4 免疫染色 -40- 2.3.5 基因表現量測定 -40- 2.3.5.1 RNA萃取 -41- 2.3.5.2 RT-PCR -42- 2.3.5.3 q-PCR -42- 三. 實驗結果以及討論 3.1 實驗動物 -45- 3.2軟骨膜前驅細胞於體外培養下之特性探討 -45- 3.2.1 組織切片分析 -45- 3.2.2 細胞型態 -45- 3.2.3 細胞標誌的表現情形 -46- 3.2.4 基因的表現情形 -46- 3.2.5 多醣體的表現情形 -46- 3.3影響軟骨分化的條件之快速篩選 -47- 3.3.1 建立篩選軟骨分化的方式 -47- 3.3.2 不同的培養液以及血清含量之條件挑選 -48- 3.3.3 不同種類以及濃度的細胞外基質之條件挑選 -49- 3.3.3.1 基質coating的均勻性及染色試驗 -50- 3.3.3.2 基質對於軟骨分化之影響 -51- 3.4 複合基質對於軟骨分化之探討 -51- 3.4.1 細胞標誌的表現情形 -52- 3.4.2 多醣體的表現情形 -53- 3.4.3 細胞型態 -53- 3.5 生長因子對於軟骨膜前驅細胞的特性以及生長之影響 -53- 3.5.1 探討生長因子對於軟骨膜前驅細胞的影響 -54- 3.5.2 建立軟骨膜前驅細胞在體外無血清的培養系統 -55- 四. 討論 -57- 五. 文獻參考 -62- 六. 附錄 -88-

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