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研究生:劉佳惠
研究生(外文):Chia-Hui Liu
論文名稱:第一型膠原蛋白誘導贅生齒牙髓幹細胞骨分化能力之探討
論文名稱(外文):Capacity of Type I Collagen on Bone-differentiation in Supernumerary Teeth Derived Dental Pulp Stem Cell
指導教授:劉念先李忠興李忠興引用關係
指導教授(外文):Nien-Hsien LiouChung-Hsing Li
口試委員:劉江川馬國興戴念梓
口試委員(外文):Jiang-Chuan LiuKUO-HSING MANiann-Tzyy Dai
口試日期:2014-05-19
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:45
中文關鍵詞:牙髓幹細胞贅生齒第一型膠原蛋白骨分化
外文關鍵詞:Dental Pulp Stem CellSupernumerary TeethType I CollagenOsteo-Differentiation
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硬骨的再生的過程可分為骨誘導、骨分化、骨質生成再到骨癒合,這些過程常需要幹細胞的參與。在骨癒合時,幹細胞分化形成骨痂可以幫助硬骨生成。近十幾年來,越來越多的研究利用第一型膠原蛋白進行幹細胞培養,研究結果顯示第一型膠原蛋白能使幹細胞骨分化後,具有高度鹼性磷酸酶活性、促進膠原蛋白的合成並增加礦化組織的形成。而本實驗使用人類贅生齒牙髓組織所培養之幹細胞結合第一型膠原蛋白,評估第一型膠原蛋白對幹細胞骨分化之影響。先以免疫螢光染色及化學染色確認贅生齒牙髓幹細胞的幹細胞特性及細胞骨分化的程度,免疫螢光染色實驗結果顯示,未分化的贅生齒牙髓幹細胞表現幹細胞特性含OCT-4、NANOG、REX-1、BMP-4和Nestin;在骨誘導一周後細胞皆持續表現中胚層標記BMP-4;而在成體細胞標記方面,骨誘導一周及兩周後細胞皆表現骨細胞標記骨鈣素,然而軟骨細胞標記SOX9卻同時表現在骨誘導兩周後未使用第一型膠原蛋白組。在茜素紅染色及von Kossa染色的結果中顯示,贅生齒牙髓幹細胞結合第一型膠原蛋白骨誘導分化後鈣離子及磷酸鹽表現增加;進一步將贅生齒牙髓幹細胞結合第一型膠原蛋白應用於顱骨缺損動物模式以評估骨修復效果,八周後以X光觀察顱骨修復效果。X光結果顯示,贅生齒牙髓幹細胞結合第一型膠原蛋白治療的組別,骨修復面積比單獨使用贅生齒牙髓幹細胞組及未治療組好。本研究的結果顯示,第一型膠原蛋白可幫助贅生齒牙髓幹細胞的分化為骨細胞及修復顱骨缺損,不僅是骨分化的改善因素同時對於贅生齒牙髓幹細胞骨分化治療的重要基質。
Bone regeneration usually needs stem cells which play a role in bone healing of osteogenesis, osteoinduction, osteoconduction and osteopromotion. Biologically, in bone healing stage, stem cells can be differentiated into components of fracture callus to help bone formation. In the last decades, there are growing studies about stem cells cultured with type I collagen. Results showed high alkaline phosphatase activity, collagen synthesis, and mineralized tissues formed. In our study, the human dental pulp stem cells from supernumerary teeth (defined DPSCs) established in our laboratory were cultured on well plate and evaluated the capacity of type I collagen on osteo-differentiation. To confirm the stemness and osteo-differentiation of DPSCs, we use immunofluorescence stain and chemical stain to quantify. The non-differentiated DPSCs expressed markers of OCT-4, NANOG, REX-1, BMP-4 and Nestin. After induction of osteo-differentiation, the early mesoderm marker, BMP4 was continuously expressed. The osteoblast marker, osteocalcin was expressed in both groups at one and two weeks after osteo-differentiation. However the chondrogenesis marker SOX9 expressed in osteo-differentiated DPSCs for two weeks without type I collagen induction. The data of Alizarin Red stain and von Kossa stain also showed positive expression in both one and two weeks with type I collagen induction. Furthermore, we grafted DPSCs with type I collagen in the rat cranial bone defects to evaluate bone repair and regeneration. Postoperative eight weeks, the animals were sacrificed and measured the cranial bone recovery by radiography. The bone repair of pulp stem cells with type I collagen group is better than other groups. The evidences of this study indicated that type I collagen is not only an improvement factor for the osteo-differentiation but also an important matrix for DPSCs based bone repair therapy.
目錄....................................................................................................................... I
圖目錄...................................................................................................................III
中文摘要...............................................................................................................V
英文摘要..............................................................................................................VII
第一章、緒論........................................................................................................1
第一節、臨床上造成骨缺損的原因.........................................................1
第二節、硬骨的胞外基質.........................................................................1
第三節、硬骨缺損的重建.........................................................................2
第四節、膠原蛋白結構及第一型膠原蛋白介紹與應用.........................3
第五節、成體幹細胞概述.........................................................................4
第六節、牙髓幹細胞的介紹.....................................................................4
第七節、研究目的.....................................................................................4
第二章、材料與方法............................................................................................6
第一節、幹細胞培養.................................................................................6
第二節、第一型膠原蛋白培養盤的製作.................................................7
第三節、硬骨誘導分化 ............................................................................7
第四節、免疫細胞螢光染色.....................................................................8
第五節、茜素紅染色 ................................................................................9
第六節、Von Kossa 染色........................................................................9
第七節、大鼠顱骨缺損實驗...................................................................10
第八節、大鼠灌流...................................................................................10
第九節、放射X光攝影 ............................................................................11
第十節、統計分析...................................................................................11
第三章、實驗結果..............................................................................................12
第四章、討論......................................................................................................14
第五章、結論......................................................................................................16
第六章、圖..........................................................................................................17
第七章、表..........................................................................................................30
第八章、參考文獻..............................................................................................31

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