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研究生：	林倩如 Lin, Chen-ju
論文名稱：	氫氧基磷灰石/膠原蛋白/透明質酸複合微粒對於間葉幹細胞骨分化之影響 Hydroxyapatite/collagen/hyaluronan composite spheres for osteogenesis of mesenchymal stem 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：	2009
畢業學年度：	97
語文別：	中文
論文頁數：	105
中文關鍵詞：	微粒、氫氧基磷灰石、骨分化、間葉幹細胞、貼附
外文關鍵詞：	hydroxyapatite, spheres, adhesion, mesenchymal stem cells, osteogenesis
相關次數：	點閱：75 下載：1
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製備含不同濃度氫氧基磷灰石(HP)的氫氧基磷灰石/膠原蛋白/透明質酸複合微粒(HC1HP、HC5HP、HC10HP、HC30HP)並利用由胎盤取得的胎盤間葉幹細胞(hPDMSC)來評估與細胞交互作用。使用於本實驗中之氫氧基磷灰石顆粒經X-ray 繞射鑑定，平均粒徑經測量約為0.3μm。hPDMSC 在低於含0.1mg/ml HP 之培養基下可正常增生。含HP 濃度越高的微粒，在濕潤環境中越不易變形。顯示HP 使基質具較好抗壓力及機械性。hPDMSC 在膠原蛋白表面貼附情形較氫氧基磷灰石良好。hPDMSC 可以貼附於含低濃度HP 之複合介面(HC1HP,HC5HP)。透過結合旋轉動態培養的方式，hPDMSC 可以貼附於含低濃度HP 之複合微粒(HC1HP, HC5HP)。在一般DMEM-10%FBS 培養下，HC10HP、HC5HP 組別相較於其他組別，鹼性磷酸酶活性在培養第7 天或第10 天時有明顯上升之情形，而在骨誘導培養基下，HC1HP、HC5HP、HC10HP 的組別，鹼性磷酸酶活性提前在第7 天時產生明顯高峰。以Real-time PCR 觀察不同介面上基因表現之差異。相較於HC0HP 之組別，在第3 天、7 天時，hPDMSC 於HC1HP之Osterix 表現量為10 倍；而在第14 天天時，Osteopontin 表現量接近15 倍，鹼性磷酸酶表現量超過5 倍。

Hydroxyapatite/ collagen/ hyaluronan composite spheres (HC0HP、HC1HP、HC5HP、HC10HP、HC30HP) containing different concentraion of hydroxyapatite were prepared and evaluate the interaction with cell by placenta derived mesenchymal stem cells(hPDMSC). The HP powder was characterized by the XRD method, and the average diameter was determined to be 0.3μm. hPDMSC proliferation was not interrupted by lower than 0.1mg/ml HP in DMEM. The spheres with higher concentration of HP (HC5HP, HC10HP, HC30HP ) maintain shape better in wet condition.hPDMSC adheres better on Col Surface when compared to HP coating surface. PDMSC can adhere on composite 2D coating with lower concentration HP. (HC1HP, HC5HP).Combining rotation method , hPDMSC can adhere on composite spheres with lower HP concentration(HC1HP, HC5HP). Alkaline phosphatase(ALP) activity significantly increased in HC10HP and HC5HP group at the 7th day culturing with DMEM. When HC10HP and HC5HP group culturing with osteogenic medium, ALP activity increased significantly t the 3rd day before normal matrix maturation stage. Moreover, comparing to HC0HP surface, hPDMSC culturing on HC1HP 、HC5HP surface, represent 10 folds osterix expression at the 3rd , 7th days, nearly 15 folds osteopontin expression and 5 folds alkaline phophatase expression at the 14th days.

中文摘要……………………………………………………………..I
英文摘要……………………………………………………………….II
目錄……………………………………………………………………..III
圖目錄……………………………………………………………....VIII
表目錄……………………………………………………………X
名詞縮寫. ……………………………………………………………XI
中英名詞對照……………………………………………………XII
第一章. 研究目的與文獻回顧
1 研究目的……………………………………….……………1
2 文獻回顧……………………………………….……………3
2.1 骨組織工程簡介…………………………………….……………3
2.1.1 組織工程……………………………………………………….3
2.1.2 骨組織工程………………………………………………………3
2.1.3 骨生醫材料…………………………………………………….4
2.1.4 骨生醫材料基本條件…………………………………………….5
2.1.5 氫氧基磷灰石…………………………………………………….6
2.1.6 透明質酸…………………………………….…………………..7
2.1.7 膠原蛋白…………………………….………………………….8
2.1.8 微粒狀基質於骨工程之應用…………………………………10
2.2 幹細胞簡介……………………………………………………….11
2.2.1 成體幹細胞…………………………………………………….11
2.2.2 間葉幹細胞…………………………………………………….11
2.2.3 胎盤組織.. ……………………………………………………..12
2.2.4 胎盤間葉幹細胞………………………………………………13
2.3 骨組織簡介…………………………………………………….15
2.3.1 骨結構…………………………………………………………...15
2.3.2 骨成分分析……………………………………………………16
2.3.3 骨細胞組成…………………………………………………….16
2.3.4 骨成型模型…………………………………………………….17
2.3.5 骨分化誘導因子………………………………………………20
2.3.6 骨分化標誌……………………………………………………24
第二章. 實驗藥品、儀器與方法
1 實驗藥品……………………………………………………27
1.1 一般常用藥品…………………………………………………27
1.2 基質成分…………………………………………………28
1.3 細胞培養、誘導分化及觀察……………………………………28
1.4 RNA 抽取、RT-PCR、PCR、電泳相關…………………………29
2 實驗儀器…………………………………………………………31
3 實驗方法…………………………………………………………33
3.1 人類胎盤間葉幹細胞(hPDMSC)之純化培……………………33
3.2 hPDMSC 之繼代培養…………………………………………36
3.3 細胞骨分化誘導及觀察…………………………………………37
3.4 hPDMSC 於含Hp 培養基之增生………………………………39
3.5 複合成分微粒之製備……………………………………………41
3.6 hPDMSC 於Hp、Col、HA 介面之貼附…………………………42
3.7 細胞培養於微粒之系統………………………………………43
3.8 冷凍組織切片……………………………………………………44
3.9 掃瞄式電子顯微鏡觀察………………………………………47
3.10 hPDMSC 於複合介面之骨分化(Alkaline phosphatase assay)…49
3.11 hPDMSC 於複合介面之骨分化(Alizarin red S)………………52
3.12 hPDMSC 於複合介面之骨分化(real-time PCR) ………………53
第三章. 複合成份基質對間葉幹細胞之骨分化影響
1 實驗構想……………………………………………………………61
2 實驗材料………………………………………………………62
3 實驗設計………………………………………………………63
3.1 HP 粉末之鑑定………………………………………………63
3.2 HP 粉體之X-ray 繞射圖譜………………………………63
3.3 hPDMSC 之繼代培養………………………………63
3.4 不同批次胎盤所取hPDMSC 骨分化潛…………….63
3.5 不同代數 hPDMSC 之骨分化潛能分析……………64
3.6 hPDMSC 於含Hp 培養基之增生…………………….64
3.7 HP/Col/HA 複合微粒製備……………………………65
3.8 複合微粒粒徑之比較…………………………………65
3.9 hPDMSC 在單一基質介面上貼附之情形…………66
3.10 hPDMSC 於複合介面貼附之情形……………………66
3.11 複合微粒培養系統……………………………………67
3.12 hPDMSC 於複合微粒貼附之情形……………………67
3.13 hPDMSC 於複合介面上鹼性磷酸酶表現………………68
3.14 hPDMSC 於複合介面之骨分化(ALP activity) ………68
3.15 hPDMSC 於複合介面之骨分化(Alizarin red) ………68
3.16 hPDMSC 於複合介面之骨分化(real-time PCR)……69
4 實驗結果……………………………………………………71
4.1 HP 粉末之鑑定……………………………………….71
4.2 hPDMSC 之繼代培養……………………………….71
4.3 不同批次胎盤所取hPDMSC 骨分化潛能……………71
4.4 不同代數 hPDMSC 之骨分化潛能分析……………72
4.5 hPDMSC 於含Hp 培養基之增生…………………72
. 4 .6 H P / C o l/HA 複合微粒製備… … … … … … … … … 7 3
4.7 複合微粒粒徑之比較……………………………………73
4.8 hPDMSC 在單一基質介面上貼附之情形…………74
4.9 hPDMSC 於複合介面貼附之情形…………………74
4.10 複合微粒培養系統…………………………………75
. 4 . 11 hPDMSC 於複合微粒貼附之情形… … … … … …75
4.12 hPDMSC 於複合介面上鹼性磷酸酶表現… … …75
4.13 hPDMSC 於複合介面之骨分化(ALP activity)…76
4.14 hPDMSC 於複合介面之骨分化(Alizarin red)…77
4.15 hPDMSC 於複合介面之骨分化(real-time PCR)77
5 結論………………………………………………………79
                                    

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校內：2011-09-07公開
校外：2012-09-07公開

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