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研究生:許芝維
研究生(外文):Chih-Wei Hsu
論文名稱:利用生醫材料促進牙髓幹細胞神經分化
論文名稱(外文):Neural Differentiation of DPSCs Promoted with Biomaterials
指導教授:楊台鴻
指導教授(外文):Tai-Horng Young
口試委員:陳羿貞
口試委員(外文):Yi-Jane Chen
口試日期:2014-07-24
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學工程學研究所
學門:工程學門
學類:綜合工程學類
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:75
中文關鍵詞:牙髓幹細胞生醫材料神經分化誘導STRO-1CD146
外文關鍵詞:DPSCsBiomaterialsNeural inductionSTRO-1CD146
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本研究利用生醫材料去促進牙髓幹細胞的神經分化,牙髓幹細胞是種多潛性幹細胞可以分化成硬骨、軟骨、脂肪及神經細胞。由於其來源容易取得,擁有的倫理爭議、個體排斥問題較小等優點,再搭配生醫材料使其達到促進分化效果。
在牙髓幹細胞神經分化的文獻整理中可以得知在模擬神經幹細胞培養的環境下其幹性以及神經分化結果較一般環境以及加藥誘導來的更好,而且也有文獻指出當代表牙髓幹細胞的標記如STRO-1表現量越高對其分化的效率更佳。因此本實驗區分成兩部分,第一部分為將牙髓幹細胞培養在可貼附以及不可貼附的高分子材料上去評估在不同天期下牙髓幹細胞中幹細胞的比例改變並找出表現量最高的材料以及培養時間。第二部分為利用在的一部分所找出幹細胞比例最高的組別並培養至聚離胺酸以及由實驗室開發的類聚離胺酸材料後以定性及定量的方法去分析其神經相關的表現。
在第一部分實驗發現經過PVA懸浮後一天牙髓幹細胞STRO-1以及CD146的表現量最高,而在第二部分發現經過懸浮後再種回TCPS、PDL及N4時使用免疫染色其神經標記如Nestin、s-III tubulin及NFM都有表現其中以N4這個材料的表現量較佳。
在電生理應用測試方面可以發現經過PVA懸浮一天後再種回TCPS、PDL及N4這三種材料上培養十天後其大部分的表現量相較於TCPS的組別來的較高而且在PVA+Induction這個系統幾乎每一顆細胞都有表現。最後在做Patch Clamp測試則發現不論是在TCPS、PDL或N4這三種材料的細胞皆沒有Na離子通。

In this study, we use biomaterials to promote the differentiation of DPSCs into neural. DPSCs is a kind of multi-potent stem cells which can differentiate into bone , cartilage, fat and neural cells. Because it is easy to access the source without many ethic problems or rejection risks. However, in this assay we will use biomaterials to promotes neural differentiation in DPSCs.
Literature review of DPSCs on neural differentiation aspect we can be learned that to simulate the Neural Stem Cells culture environment DPSCs’ stemness and neural differentiation ability will be better than the normal and general induction environment. Therefore, there is another literature shows that when the STRO-1 marker( representatives as DPSCs stem cell marker) has higher expression that means it will have better differentiation ability. Therefore, in this study we will divided it into two parts, the first part is to culture DPSCs in polymer materials which can be attached or not and at a specific culture time to assess the highest proportion of stem cells. Based on part one, second part is to culture the high proportion of DPSCs into poly-D-lysine and poly-D-lysine like material which developed by the laboratory to qualitative and quantitative analysis the nerve -related performance.
In the part one experiment we found that when DPSCs during one day floated on PVA the expression of STRO-1 and CD146 was the highest. At the second part DPSCs reseeded on TCPS, PDL and N4 and from the immunofluorescence result we can found the neural marker such as Nestin, s-III tubulin and NFM has a performance on almost every materials and N4 was the best.
The neural function test of PVA one day on part one can be better than on TCPS. Moreover, when add induction medium on the PVA system almost every cell has excellent performance. Finally doing Patch Clamp found that there are no Sodium ion channel on all of the materials.


中文摘要 i
Abstract ii
圖目錄 vii
第一章 緒論 1
第二章 文獻回顧 3
2.1 牙髓幹細胞(Dental Pulp Stem Cells, DPSCs) 3
2.1.1 一般培養環境 3
2.1.2 加藥誘導方法 4
2.1.3 成球加藥誘導方法 5
2.2 細胞成球方法 5
2.3 聚乙烯醇 7
2.4 幾丁聚醣 8
2.5 聚離胺酸 9
2.6 仿聚離胺酸 10
第三章 實驗材料與方法 11
3.1 實驗理論與架構 11
3.2 試劑配製 12
3.2.1 磷酸緩衝溶液 (Phosphate Buffer Solution) 12
3.2.2 培養液 (Dulbecco’s Modified Eagle Medium low glucose) 12
3.2.3 Trypsin-EDTA 溶液 13
3.3實驗方法 13
3.3.1 細胞培養 13
3.3.2 聚乙烯醇 (Polyvinyl Alcoho) 基材製備 14
3.3.3 幾丁聚醣 (Chitosan) 基材製備 14
3.3.4 聚離胺酸 (Poly-D-lydine) 基材製備 14
3.3.5 仿聚離胺酸 Poly-D-lydine like) 基材製備 14
3.3.6 神經分化誘導 15
3.3.7 流式細胞儀分析 16
3.3.8 免疫螢光染色( Immunofluorence) 16
3.3.9 基因表現 (PCR) 16
3.3.10 神經細胞功能測試 18
3.3.10.1突觸素I 蛋白(Phospho-Synapsin I) 磷酸化程度評估 18
3.3.10.2電生理測試(膜片箝制, Patch Clamp) 19
3.3.10.2.1藥品與溶液 19
3.3.10.2.2全細胞紀錄 19
第四章 實驗結果 20
4.1 牙髓幹細胞 20
4.2 牙髓幹細胞在不同材料上型態表現 20
4.3 牙髓幹細胞的幹性標記表現 22
4.4 牙髓幹細胞的神經標記表現 24
4.5 牙髓幹細胞的神經分化效率 27
4.5.1免疫螢光染色定性分析 27
4.5.2 基因方面(PCR)定量分析 28
4.6 牙髓幹細胞的神經功能測試 29
4.6.1突觸素I蛋白(Phospho-Synapsin I) 磷酸化程度評估 29
4.6.2電生理測試(膜片箝制,Patch Clamp) 29
第五章 討論 30
5.1牙髓幹細胞幹性 30
5.2 牙髓幹細胞的神經分化 30
5.3 牙髓幹細胞在不同材料上培養的結果討論 31
5.3.1牙髓幹細胞的型態 31
5.3.2 牙髓幹細胞的幹細胞比例 31
5.3.3 牙髓幹細胞的神經marker 32
5.4 牙髓幹細胞的神經分化誘導方式及結果 34
5.4.1 神經分化誘導方式 34
5.4.2 神經分化誘導結果 35
5.4.2.1免疫螢光染色 35
5.4.2.2 基因表現(PCR) 35
5.4.2.3突觸素I蛋白(Phospho-Synapsin I) 磷酸化程度評估 35
5.4.2.4膜片箝制(Patch Clamp)電生理測試 36
第六章 結論 37
第七章 參考文獻 38
第八章 圖表 40




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