楊台鴻臺灣大學:醫學工程學研究所鍾明均Chung, Min-ChunMin-ChunChung2010-05-182018-06-292010-05-182018-06-292009U0001-2107200900473600http://ntur.lib.ntu.edu.tw//handle/246246/183702人類羊膜是一天然性生物材料，目前已有臨床報告指出，羊膜可被成功應用為貼附材料以促進傷口癒合、眼角膜重建等；隨著幹細胞研究的發展，利用組織工程修復口腔組織缺損已不是遙不可及的夢想，近年來已有研究證實牙根尖乳突細胞群在適當的誘導下能表現多重分化的潛能，因此，本研究的目的在探討羊膜作為口腔細胞生長基材的情形，以及羊膜對於牙根尖乳突細胞生長及功能的影響，以評估將羊膜應用於口腔內組織修復的可能性。 為探討羊膜是否適合口腔細胞之附著及生長，我們分別以牙根尖乳突細胞、牙齦纖維母細胞、牙周韌帶纖維母細胞、牙髓細胞與骨髓間葉幹細胞等五種細胞進行基本測試，測試各種細胞在鋪植後不同時間點（1、3、5、7天）的MTT活性，比較羊膜基材用於口腔細胞培養的適合性。將各種細胞培養在經去細胞處理的羊膜基底層與實質層上，對其貼附、活性及形態做探討，從結果中發現，牙根尖乳突細胞在羊膜基材上的貼附與活性並不如tissue culture polystrene（TCPS），牙髓細胞與骨髓間葉幹細胞在生長活性上也有類似的結果，但對於牙齦纖維母細胞及牙周韌帶纖維母細胞來說，羊膜是有利於細胞生長的基材。 本研究對於培養自發育中第三大臼齒根尖牙乳突部位之細胞進行初步鑑定，利用STRO-1與CD146抗體做免疫螢光染色及流式細胞儀分析，探討牙根尖乳突細胞是否具有間葉幹細胞的特性，結果顯示部分牙根尖乳突細胞群對此兩種抗體的表現為陽性，可說明本研究所培養測試之人類牙根尖乳突細胞為具有間葉細胞特性的前驅細胞。在促進細胞基質礦化之培養液中，人類牙根尖乳突細胞的鹼性磷酸酶活性表現隨培養時間增長而上升，顯示其具有類似成骨細胞分化的基本特性；而相對於TCPS，生長在羊膜基材上的牙根尖乳突細胞都具有較高的ALP活性，細胞外基質礦化程度也較強，且有提早促進COL I（collagen type I）、Cbfa1（Core-binding factor-1）、ALP（alkaline phospatase）及OC（osteocalcin）基因表現的效應。 本研究更進一步利用ERK1/2抑制劑（U0126）探討牙根尖乳突細胞在羊膜上之分化促進是否與ERK1/2訊息傳導路徑有關，結果証明牙根尖乳突細胞的分化確實與ERK1/2有關，但關於羊膜基質對於細胞的成骨分化能力之促進作用，ERK或許並不是最主要的角色。Human amniotic membrane (AM) is a nature biomaterial which has been successfully used in cornea reconstruction and as a biological dressing for wounds. Recently, it was reported that apical papilla cells were multipotent and their ability of differentiating into specific lineage could be induced in an appropriate environment. Therefore, the purpose of this study was to investigate the possibility of applying human amnions for oral tissue repair by evaluating the growth and function of human apical papilla cells cultured in the de-cellular human amniotic matrix. In addition to apical papilla cells, several kinds of dental mesenchymal cells were used to check the feasibility of de-cellular AM matrix for cellular adhesion and growth, including dental pulp cells, gingival fibroblasts, periodontal ligament fibroblasts, and bone marrow mesenchymal stem cells. The apical papilla cells were cultured from human apical papilla of developing third molar extracted due to orthodontic reason. The dental progenitor cells in the mixed population cultured from apical papilla was characterized by using anti- STRO-1 and anti- CD146 antibody for immunofluorescence stain and flow cytometry. The osteogenic differentiation of apical papilla cells grown on different substrates (tissue culture plate (TCPS), basement membrane of AM, and collagenous matrix of AM) was investigated by measuring the alkaline phosphotase activity and extra-cellular matrix mineralization revealed by von Kossa stain. The mRNA expressions of osteoblast-related genes (COL I, Cbfa1, ALP, and OC) were checked by using RT-PCR technique. Protein expression and phosphorylation of RUNX-2 were detected via the procedures of imunoprecipitation and western blot. Moreover, we used U0126, an inhibitor of ERK1/2 pathway, to check if the ERK 1/2 pathway is involved in the osteogenic differentiation of apical papilla cells grown on AM. The result showed that the adhesion and proliferation of apical papilla cells cultured on amniotic membrane were not as good as those on TCPS. The similar difference between amniotic membrane and TCPS for cellular growth was noted for dental pulp cells and bone marrow mesenchymal stem cells. However, amniotic membrane was a good matrix to support the growth of gingival fibroblasts and periodontal ligament fibroblasts. The osteoblastic phenotype of human apical papilla cells was demonstrated by their progressive increase of ALP activity and extracellular matrix mineralization in long-term culture. Compared to TCPS, AM could promote osteogenic differentiation of apical papilla cells. The mRNA expression of COL I, Cbfa1, ALP, and OC was also up-regulated in apical papilla cells grown on AM. Moreover, the osteogenic differentiation of apical papilla cells, either on TCPS or on AM, was significantly down-regulated in the presence of U0126. It implied that ERK1/2 signaling pathway was activated in the osteogenic differentiation of apical papilla cells, followed by up-regulating the expression and phosphorylation of RUNX-2, subsequently increasing ALP activity and mineralized matrix deposition. However, the promoting effect of AM on the osteogenic differentiation of apical papilla cells may not directly involve the ERK1/2 signaling pathway.摘要 ibstract iii錄 v目錄 ix目錄 xi一章 緒論 1-1 研究背景 1-1-1 牙周組織與缺損 1-1-2 牙周修復與牙齒再生 2-1-3 組織工程（tissue engineering）原理 3-2 研究目的 4二章 文獻回顧 5-1 牙齒發育及牙囊組織的特徵 5-2 牙根尖乳突細胞的特性 6-3 細胞分化與成骨指標蛋白的基因表現 8-3-1 第一型膠原蛋白（Collagen type I，COL I） 9-3-2 Cbfa1/Runx2（Core-binding factor-1/Runt-related transcription factor-2） 9-3-3 鹼性磷酸酶（Alkaline phosphatase，ALP） 10-3-4 骨鈣蛋白（Osteocalcin，OC） 10-4 人類羊膜的組織形態 10-5 人類羊膜於體外培養實驗的模式 11-6 應用羊膜於促進傷口癒合的模式 12三章 實驗材料與方法 15-1 細胞培養（cell culture） 15-1-1 人類牙根尖乳突細胞（Human Apical papilla cells，DF） 15-1-2 人類牙周韌帶纖維細胞（Human Periodontal Ligament Fibroblasts，PF） 16-1-3 人類牙齦纖維細胞（Human Gingival Fibroblasts，GF） 16-1-4 人類牙髓細胞（Human Dental Pulp cells，DP） 16-1-5 人類骨髓幹細胞（Human Bone Marrow Stem cell，BMSC） 17-2 去細胞羊膜之配製 17-3 礦化培養液（Mineralizing Medium）之配製 18-4 細胞生長測試（MTT assay） 18-5 免疫螢光染色法（immunofluorescence stain） 19-5-1 STRO-1與CD146染色 19-5-2 肌動蛋白細胞骨架染色（Actin Cytoskeleton Staining） 20-5-3 Hoechst 33258染色 20-6 流式細胞儀分析（Flow cytometry） 21-7 促進基質礦化實驗之成骨基因表現 21-7-1 RNA萃取 21-7-2 反轉錄聚合酶連鎖反應（Reverse Transcription-Polymerase Chain Reaction，RT-PCR） 22-7-3 瓊脂凝膠電泳分析（Agarose gel electrophoresis） 23-8 鹼性磷酸酶活性（Alkaline Phosphatase Activity）測定 23-9 鈣化節點染色法（von Kossa stain） 25-10 西方墨點法（Western Blot） 25-10-1 細胞內蛋白質分離 26-10-2 免疫沉澱（immunoprecipitation，IP） 26-10-3 西方墨點法（Western Blot） 27-11 統計分析 28四章 結果 29-1 MTT活性測試 29-1-1 人類牙根尖乳突細胞培養於TCPS及羊膜基材之生長情形 29-1-2 人類牙周韌帶纖維母細胞培養於TCPS及羊膜基材之生長情形 29-1-3 人類牙齦纖維母細胞培養於TCPS及羊膜基材之生長情形 30-1-4 人類牙髓細胞培養於TCPS及羊膜基材之生長情形 30-1-5 人類骨髓間葉幹細胞培養於TCPS及羊膜基材之生長情形 30-2 牙根尖乳突細胞形態與鑑定 30-2-1 牙根尖乳突細胞形態觀察 30-2-2 牙根尖乳突細胞鑑定 31-3 牙根尖乳突細胞培養於TCPS與羊膜的情形 31-3-1 細胞貼附的形態 31-3-2 Hoechst33258染色結果 32-4 成骨基因表現 33-4-1 COL I 基因表現 33-4-2 Cbfa1 基因表現 33-4-3 ALP基因表現 34-4-4 OC基因表現 34-5 鹼性磷酸酶活性測定 34-6 鈣化節點染色法觀察 35-7 Cbfa1/Runx2蛋白質表現 36-8 U0126作用的影響 36-8-1 不同濃度之U0126對於牙根尖乳突細胞生長活性之測試 37-8-2 不同濃度之U0126對於牙根尖乳突細胞之鹼性磷酸酶活性測試 37-8-3 U0126在不同材料上對ERK1/2磷酸化的影響 37-8-4 U0126在不同材料上對成骨化基因的影響 38-8-5 U0126在不同材料上之鹼性磷酸酶活性差異 38-8-6 U0126在不同材料上之鈣化節點染色法 39-8-7 U0126在不同材料上之Cbfa1/Runx2蛋白磷酸化的影響 39五章 討論 40-1 牙根尖乳突細胞形態 40-2 牙根尖乳突細胞鑑定 40-3 羊膜對牙根尖乳突細胞生長的影響 41-4 促進牙根尖乳突細胞基質礦化的研究 42-5 人類羊膜對牙根尖乳突細胞成骨分化的影響 45-5-1 COL I（collagen type I） 46-5-2 Cbfa1/Runx2（Core-binding factor-1/Runt-related transcription factor-2） 46-5-3 ALP（alkaline phosphase） 47-5-4 OC（osteocalcin） 48-6 牙根尖乳突細胞鈣化沉積物的產生 49-7 羊膜影響牙根尖乳突細胞分化能力的機制 50六章 結論 53七章 未來研究方向 55考文獻 56錄（表與圖） 66application/pdf4605839 bytesapplication/pdfen-US人類羊膜牙根尖乳突細胞成骨分化human amniotic membraneapical papilla cellsosteogenic differentiationthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/183702/1/ntu-98-R96548016-1.pdf