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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳敏慧 | |
| dc.contributor.author | Hsin-Hua Lin | en |
| dc.contributor.author | 林欣樺 | zh_TW |
| dc.date.accessioned | 2021-06-08T06:57:37Z | - |
| dc.date.copyright | 2009-09-15 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-07-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25960 | - |
| dc.description.abstract | 牙髓幹細胞( dental pulp stem cells, DPSC )取自於牙髓組織，是一具有高度增生速率及分化潛能的前驅細胞。我們利用中胚層間葉幹細胞( mesenchymal stem cells, MSC )的表面抗原CD146和STRO-1將這類細胞分離出來，培養在促進基質礦化之培養基中進行牙髓幹細胞的分化。
透過互補核苷酸基因微陣列( cDNA microarray )基因分析，牙髓幹細胞在分化成類牙本質母細胞( odontoblast-like cells )的過程中，除了一些跟細胞分化有相關的基因會向上調控，例如ALP，BMP2等之外，從基因功能性的圖譜顯示metallothionein mRNA的表現與細胞礦化作用有相關，其中又以MT1F的表現最為明顯。因此，我們認為MT1F在調控牙髓幹細胞分化的過程中扮演了重要的角色。此外，其他研究亦有報告，由metallothionein所轉錄出來的蛋白質-金屬硫蛋白( metallothionein, MT )參與了細胞內的多項調控，包括了細胞的生長、抗氧化、結合重金屬。 由鹼性磷酸酶( Alkaline Phosphatase, ALP )和基質礦化小體( Alizarin Red S, ARS )染色以及即時定量聚合酶連鎖反應( Real-Time PCR ) 的實驗結果顯示 : 利用濃度50 μM-200 μM硫化鋅(ZnCl2)誘導內生性的metallothionein 1F(MT1F)的過度表現可以促進細胞礦化的生成和dentin – sialophosphoprotein ( DSPP )、osteocalcin ( OCN )基因的表現量增加，尤其以ZnCl2濃度為100 μM最為顯著。因此，本研究結果證實，metallothionein 1F的表現確實可以促進牙本質母細胞生成以及縮短礦化作用表現的時間，我們推論MT1F是一個被新發現可促進牙髓幹細胞分化的因子。 | zh_TW |
| dc.description.abstract | Post-natal human dental pulp stem cells (DPSCs) are derived from dental pulp tissue. Several reports have been demonstrated that dental pulp stem cells have high proliferation rate and multipotent differentiation capacity. By using mesenchymal stem cells surface markers – STRO-1 and CD146, DPSCs were sorted by flow cytometry and were cultured in the basal medium till cells were confluence. The medium was then changed with mineralization enhancing medium (inductional medium) for inducing DPSCs differentiation.
After cDNA microarray analysis, some genes related to cell differentiation such as ALP and BMP2 were found to be upregulated. In addition, ingenuity pathway analysis (IPA) data revealed that metallothionein mRNA expression was associated with cell mineralization, especially the expression of metallothionein 1F (MT1F) gene. Thus, we hypothesized that metallothionein 1F may play an important role in DPSCs differentiation. According to previous report, metallothionein (MT) protein is encoded from metallothionein mRNA and it can mediate a lot of intracellular regulation such as cell proliferation, differentiation and regulation of essential metals and protection of cells against oxidative injury. In this study, the effect of metallothionein on DPSCs differentiation was examined. From the results of alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining and RT-PCR as well as real-time PCR analysis, it was found that cells pretreated with 50 μM-200 μM ZnCl2, were demonstrated with overexpression of endogenous metallothionein. Thus, odontoblasts related genes – DSPP and OCN genes expression were upregulated and cells were shown with better mineralization and differentiation potential than those in the control group. The results were enhanced especially in the 100 μM treated group. Our results indicated that overexpression of metallothionein 1F gene can promote DPSCs to be differentiated into odontoblasts, and shorten the time for mineralization and differentiation. In conclusion, we found metallothionein 1F is a novel factor that can enhance DPSCs differentiation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T06:57:37Z (GMT). No. of bitstreams: 1 ntu-98-R96450014-1.pdf: 7365712 bytes, checksum: 8e1393b410e5192191fd9ebc9e825c65 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 目錄……………………………………………………………………………………I
圖次目錄………………………………………………………………………………IV 表次目錄………………………………………………………………………………V 中文摘要………………………………………………………………………………VI 英文摘要………………………………………………………………………………VII 第一章 引言………………………………………………………………………...1 1. 牙齒的發育........................................................................................................1 2. 幹細胞的來源…………………………………………………………………2 3. 牙髓幹細胞的發展……………………………………………………………3 4. 促進牙髓幹細胞分化的基因…………………………………………………6 5. 牙髓幹細胞與組織工程………………………………………………………6 6. 互補核苷酸基因微陣列………………………………………………………7 7. 金屬硫蛋白.…………………………………………………………………...9 第二章 實驗目的…………………………………………………………………..12 第三章 材料與方法………………………………………………………………..13 1. 細胞培養……………………………………………………………………..13 2. 牙髓幹細胞的鑑定…………………………………………………………..13 3. 細胞礦化生成培養液之製備………………………………………………..13 4. 脂肪生成培養液之製備……………………………………………………..14 5. 互補核苷酸基因微陣列…………………………………………….………..14 6. 不同濃度之ZnCl2配製……………………………………………………...14 7. 細胞活性測試.………………………………………………………………..15 8. 鹼性磷酸酶染色與觀察……………………………………………………...15 9. 細胞基質礦化小體染色……………………………………………………...16 10. 鹼性磷酸酶活性測試………………………………………………………...17 11. 基因表現分析………………………………………………………………...18 12. 統計分析……………………………………………………………………...22 第四章 結果………………………………………………………………………...23 1. 牙髓細胞型態觀察…………………………………………………………...23 2. 流式細胞儀分選牙髓幹細胞………………………………………………...23 3. 細胞培養於一般培養液及礦化培養液之細胞型態觀察.…………………..24 4. RT-PCR分析牙髓幹細胞分化成牙本質母細胞之基因表現………………24 5. cDNA microarray分析………………………………………………….……25 6. 細胞活性測試………………………………………………………………..26 7. 鹼性磷酸酶染色的觀察……………………………………………………..26 8. 鹼性磷酸酶的活性測定……………………………………………………..27 9. ARS礦化小體染色的觀察………………………………………………….27 10. real-time PCR分析誘導後基因的表現……………………………………..28 第五章 討論………………………………………………………………………..30 1. 牙髓幹細胞的分離以及細胞型態…………………………………………..30 2. 牙髓幹細胞分化的能力.…………………………………………………….31 3. 分化過程中向上調控的基因………………………………………………..32 4. metallothionein mRNA的誘導……………………………………………...35 5. 過度表現metallothionein對牙髓幹細胞分化的影響……………………...36 6. Metallothionein, MT金屬硫蛋白的應用.…………………………………...38 第六章 結論………………………………………………………………………..40 第七章 未來研究方向……………………………………………………………..42 參考資料……………………………………………………………………………..69 | |
| dc.language.iso | zh-TW | |
| dc.title | 利用基因微陣列探討牙髓幹細胞分化之基因表現 | zh_TW |
| dc.title | cDNA Microarray Analysis of Genes Expression in Dental Pulp Stem Cells Differentiation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林劭品,江俊斌 | |
| dc.subject.keyword | 牙髓幹細胞,牙本質母細胞,基因微陣列,金屬硫蛋白,細胞分化, | zh_TW |
| dc.subject.keyword | dental pulp stem cells(DPSC),odontoblasts,cDNA microarray,metallothionein 1F,cell differentiation, | en |
| dc.relation.page | 75 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2009-07-16 | |
| dc.contributor.author-college | 牙醫專業學院 | zh_TW |
| dc.contributor.author-dept | 口腔生物科學研究所 | zh_TW |
| 顯示於系所單位： | 口腔生物科學研究所 | |
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