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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭景暉(Jiiang-Huei Jeng),張曉華(Hsiao-Hua Chang) | |
| dc.contributor.author | Wan-Chun Hsieh | en |
| dc.contributor.author | 謝宛君 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:55:47Z | - |
| dc.date.available | 2021-08-30 | |
| dc.date.copyright | 2018-08-30 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-07-27 | |
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J Huazhong U Sci, 27(1), 44-47. doi:10.1007/s11596-007-0113-2 Zhou, X., Zhang, Z., Feng, J. Q., Dusevich, V. M., Sinha, K., Zhang, H., . . . de Crombrugghe, B. (2010). Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice. Proc Natl Acad Sci U S A, 107(29), 12919-12924. doi:10.1073/pnas.0912855107 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71153 | - |
| dc.description.abstract | 實驗目的:本實驗目的是透過研究osterix（osx），巢蛋白(nestin)，波形蛋白(vimentin)，結締組織生長因子（CTGF），N-鈣粘蛋白(N-cadherin)，牙骨質蛋白1（CEMP1），牙骨質附著蛋白（CAP）和纖溶酶原激活抑製劑-1（PAI-1）等分化相關標誌物的表現，探索TGF-β1誘導人類牙根尖細胞分化的潛力。並且探索其相關的訊號傳遞途徑。
實驗方法 : 將人類牙根尖細胞暴露於不同濃度（0, 0.5, 1, 5, 10, 25 ng / ml）的TGF-β1下24小時，利用分化相關標誌物如osx，nestin，vimentin，CTGF，N-cadherin，CEMP1，CAP和PAI-1透過反轉錄聚合酶連鎖反應(RT-PCR)，西方點墨法(western blot)以及免疫螢光染色來看其影響基因或蛋白的表現為何。此外，使用SB431542（ALK5 / Smad2抑制劑）的預先處理來研究TGF-β1誘導人類牙根尖細胞的分化作用是否透過Smad依賴性（經典）訊號途徑傳遞。 實驗結果 : 暴露於TGF-β1 24小時的人類牙根尖細胞沒有明顯的形態改變。當加入TGF-β1時，osx，nestin，vimentin，CTGF，N-cadherin，CEMP1，CAP和PAI-1的基因和/或蛋白質表現增加。此外，加入1, 2.5 μM SB431542後減少了TGF-β1誘導的nestin，vimentin，CTGF，N-cadherin和PAI-1的表現。然而，加入抑制劑SB431542並未抑制osx，CEMP1和CAP的表現。 結論 : TGF-β1可能具有誘導人類牙根尖細胞多種分化潛能的能力，例如骨生成，牙本質形成，牙骨質生成，以及影響細胞外基質生成轉換。由TGF-β1誘導的nestin，vimentin，CTGF，N-cadherin和PAI-1的表現是透過ALK5 / Smad2信號傳遞路徑。然而，由TGF-β1誘導的osx，CEMP1和CAP的表現則不是經由此路徑傳遞。TGF-β1及牙根尖細胞應具有應用在臨床再生性牙髓治療的潛力。 | zh_TW |
| dc.description.abstract | Aim : The aim of the study is to investigate the differentiation potentials of human apical papilla cells induced by TGF-β1 by analyzing the expressions of differentiation related markers, including osterix (osx), nestin, vimentin, connective tissue growth factor (CTGF), N-cadherin, cementum protein 1 (CEMP1), cementum attachment protein(CAP), and plasminogen activation inhibitor-1(PAI-1). The signaling pathway conveying the effects was also explored.
Materials and methods: Primary-cultured human apical papilla cells were treated with different concentrations (0, 0.5, 1, 5, 10, 25 ng/ml) of TGF-β1 for 24 hours. The expressions of osx, nestin, vimentin, CTGF, N-cadherin, CEMP1, CAP, and PAI-1 were evaluated through RT-PCR, western blot, and immunofluorescence. In addition, pretreatment of SB431542 (as a specific ALK5/Smad2 inhibitor) was used to investigate the Smad-dependent (canonical) pathway in TGF-β1-induced effects on human apical papilla cells. Results: Human apical papilla cells exposed to TGF-β1 for 24 hours showed no obvious morphology change. The mRNA and/or protein expressions of osx, nestin, vimentin, CTGF, N-cadherin, CEMP1, CAP, and PAI-1 showed increased when adding TGF-β1. Besides, pretreatment of 1and 2.5 µM SB431542 reduced TGF-β1 stimulated-expressions of nestin, vimentin, CTGF, N-cadherin, and PAI-1. However, the expressions of osx, CEMP1, and CAP were not inhibited by adding inhibitor SB431542. Conclusions : TGF-β1 may be capable to induce various differentiation potentials of human apical papilla cells such as osteogenesis, odontogenesis and cementogenesis, and to modulate the turnover of extracellular matrix. The expressions of nestin, vimentin, CTGF, N-cadherin, and PAI-1 induced by TGF-β1 were conveyed through ALK5/Smad2 signaling pathway. However, the expressions of Osx, CEMP1, and CAP induced by TGF-β1 were not. The information may benefit the development of new strategies for regenerative endodontics. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:55:47Z (GMT). No. of bitstreams: 1 ntu-107-R04422010-1.pdf: 12144634 bytes, checksum: f0a6390dda9cc20f1121300527e32b74 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員審定書 i
謝誌 ii 中文摘要 iii Abstract v Chapter I. Literature review 1 1.1 Regenerative endodontics 1 1.2 Introduction of stem cells from apical papilla 3 1.3 Introduction of transforming growth factor beta 7 1.3.1 TGF-β superfamily 7 1.3.2 TGF-β isoforms and their peptide structure 9 1.3.3 Receptors and signaling pathways of TGF-β 10 1.3.3.1 Canonical signal transduction pathways of TGF-β 11 1.3.3.2 Non-canonical signal transduction pathways of TGF-β 12 1.3.4 The effect of TGF-β1 on dental cells 13 1.4 Differentiation related markers 13 1.4.1 Osterix (OSX, SP7) 13 1.4.2 Nestin 14 1.4.3 Vimentin 16 1.4.4 N-cadherin 17 1.4.5 Connective tissue growth factor 18 1.4.6 Cementum protein 1 (CEMP1) 19 1.4.7 Cementum attachment protein (CAP) 20 1.4.8 Plasminogen activator inhibitor-1 (PAI-1) 21 Chapter II. The purpose of the study 23 Chapter III. Materials and methods 24 3.1 Materials 24 3.2 Culture of human apical papilla cells 26 3.3 Reverse transcription-polymerase chain reaction(RT-PCR) 26 3.3.1 Total RNA isolation 27 3.3.2 RNA quantification 29 3.3.3 Reverse transcription (RT) 29 3.3.4 Polymerase chain reaction (PCR) 30 3.4 Western blot 31 3.4.1 Protein extraction 32 3.4.2 Protein quantification 33 3.4.3 Sodium dodecyl sulfate-polyarylamide gel electrophoresis (SDS-PAGE) 34 3.4.4 Transfer gel 35 3.4.5 Blocking and antibody hybridization 35 3.4.6 Chemiluminescence photography 36 3.5 Immunofluorescent (IF) observation 36 Chapter IV. Results 40 4.1 Morphological change of human apical papilla cells 40 4.2 Effects of different concentrations of TGF-β1 on human apical papilla cells 40 4.2.1 Expressions of ALK5 mRNA and p-Smad2 protein in TGF-β1 exposed cells 40 4.2.2 Expressions of osterix mRNA and protein in TGF-β1 exposed cells 41 4.2.3 Expressions of nestin mRNA, protein and immunofluorescence staining in TGF-β1 exposed cells 41 4.2.4 Expression of vimentin protein in TGF-β1 exposed cells 41 4.2.5 Expressions of N-cadherin mRNA and protein in TGF-β1 exposed cells 42 4.2.6 Expressions of CTGF mRNA and protein in TGF-β1 exposed cells 42 4.2.7 Expressions of mRNA of CEMP1 and mRNA and protein of CAP in TGF-β1 exposed cells 43 4.2.8 Expression of PAI-1 mRNA in TGF-β1 exposed cells 43 4.3 Effect of TGF-β1 with ALK5-Smad2 inhibitor SB431542 on human apical papilla cells 43 4.3.1 Effect of TGF-β1 on ALK5 and p-Smad2 expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 44 4.3.2 Effect of TGF-β1 on osterix expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 44 4.3.3 Effect of TGF-β1 on nestin expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 44 4.3.4 Effect of TGF-β1 on vimentin expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 45 4.3.5 Effect of TGF-β1 on N-cadherin expression with ALK-Smad2 inhibitor SB431542 in human apical papilla cells 45 4.3.6 Effect of TGF-β1 on CTGF expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 45 4.3.7 Effect of TGF-β1 on CEMP1 and CAP expressions with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 46 4.3.8 Effect of TGF- β1 on PAI-1 expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 46 Chapter V. Discussion 47 Chapter VI. Conclusion 55 References 56 Appendix App.1. A schematic representation of TGF-β superfamily. 68 App.2. A schematic representation of TGF-β different forms occurring during synthesis, secretion, and activation. 69 App.3. TGF-β1 associated intracellular canonical and non-canonical signaling pathways. 70 App.4. TGF-β1 signaling pathways. 71 App.5. Hypothesis of the study 72 App.6. A predicted signaling flow chart to decipher how TGF-β1 affects the expression of each marker. . 73 Tables Table 1. PCR primer sequences 74 Table 2. Protocol of western blot protein extraction buffer 75 Table 3. Protocol for SDS-PAGE 75 Table 4a. Protocol for western blot SDS-PAGE running buffer 77 Table 4b. Protocol for western blot SDS-PAGE transfer buffer 77 Table 4c. Protocol for western blot tween TBS 77 Table 5. Western blot primary antibodies. 78 Figures Figure 1. Apical papilla (I-Hua Wu, 2011) 79 Figure 2A. Morphological change of human apical papilla cells (100x magnification) 80 Figure 2B. Morphological change of human apical papilla cells (100x magnification) 81 Figure 3. Expressions of ALK5 mrna and p-Smad2 protein in TGF-β1 exposed cells 82 Figure 4. Expressions of osterix mRNA and protein in TGF-β1 exposed cells 83 Figure 5. Expressions of nestin mRNA and protein in TGF-β1 exposed cells 84 Figure 6. Expressions of vimentin protein in TGF-β1 exposed cells 85 Figure 7. Expressions of N-cadherin mRNA and protein in TGF-β1 exposed cells 86 Figure 8. Expressions of CTGF mRNA and protein in TGF-β1 exposed cells 87 Figure 9. Expressions of mRNA of CEMP1 and mRNA and protein of CAP in TGF-β1 exposed cells 88 Figure 10. Expressions of PAI-1 mrna in TGF-β1 exposed cells 89 Figure 11. Effect of TGF-β1 on ALK5 and p-Smad2 expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 90 Figure 12. Effect of TGF-β1 on osterix expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 91 Figure 13. Effect of TGF-β1 on nestin expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 92 Figure 14. Effect of TGF-β1 on vimentin expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 93 Figure 15. Effect of TGF-β1 on N-cadherin expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 94 Figure 16. Effect of TGF-β1 on CTGF expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 95 Figure 17. Effect of TGF-β1 on CEMP1 and CAP expressions with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 96 Figure 18. Effect of TGF-β1 on PAI-1 expression with ALK5-Smad2 inhibitor SB431542 in human apical papilla cells 97 Figure 19A. Immunofluorescence staining result. Effect of TGF-β1 on expression nestin in human apical papilla cells(100X). 98 Figure 19B. Immunofluorescence staining result. Effect of TGF-β1 on expression nestin in human apical papilla cells(400X). 99 Figure 20A. Immunofluorescence staining result. Effect of TGF-β1 on expression vimentin in human apical papilla cells(100X). 100 Figure 20B. Immunofluorescence staining result. Effect of TGF-β1 on expression vimentin in human apical papilla cells(400X). 101 Figure 21A. Immunofluorescence staining result. Effect of TGF-β1 on expression N-cadherin in human apical papilla cells(100X). 102 Figure 21B. Immunofluorescence staining result. Effect of TGF-β1 on expression N-cadherin in human apical papilla cells(400X). 103 Figure 22A. Immunofluorescence staining result. Effect of TGF-β1 on expression of CTGF human apical papilla cells(100X). 104 Figure 22B. Immunofluorescence staining result. Effect of TGF-β1 on expression of CTGF in human apical papilla cells(400X). 105 Figure 23A. Immunofluorescence staining result. Effect of TGF-β1 on expression CAP in human apical papilla cells(100X). 106 Figure 23B. Immunofluorescence staining result. Effect of TGF-β1 on expression CAP in human apical papilla cells(400X). 107 Figure 24A. Effect of 10 ng/ml TGF-β1 with pretreatment 1, 2.5 µm SB431542 on expression of nestin in human apical papilla cells(100X). 108 Figure 24B. Immunofluorescence staining result. Effect of 10 ng/ml TGF-β1 with pretreatment 1, 2.5 µm SB431542 on expression nestin in human apical papilla cells (400X). 109 Figure 25A. Effect of 10 ng/ml TGF-β1 with pretreatment 1, 2.5 µm SB431542 on expression N-cadherin in human apical papilla cells(100X). 110 Figure 25B. Immunofluorescence staining result. Effect of 10 ng/ml TGF-β1 with pretreatment 1, 2.5 µm SB431542 on expression N-cadherin in human apical papilla cells(400X). 111 Figure 26A. Effect of 10 ng/ml TGF-β1 with pretreatment 1, 2.5 µm SB431542 on expression CTGF in human apical papilla cells(100X). 112 Figure 26B. Immunofluorescence staining result. Effect of 10 ng/ml TGF-β1 with pretreatment 1, 2.5 µm SB431542 on expression CTGF in human apical papilla cells(400X). 113 | |
| dc.language.iso | en | |
| dc.subject | 轉化生長因子β1 | zh_TW |
| dc.subject | 經典訊號傳遞路徑 | zh_TW |
| dc.subject | 抑制劑 | zh_TW |
| dc.subject | 分化相關標誌物 | zh_TW |
| dc.subject | 人類牙根尖細胞 | zh_TW |
| dc.subject | ALK5/Smad2 signaling pathway | en |
| dc.subject | human apical papilla cells | en |
| dc.subject | differentiation related markers | en |
| dc.subject | SB431542 | en |
| dc.subject | inhibitor | en |
| dc.subject | TGF-β1 | en |
| dc.title | TGF-β1對於人類牙根尖細胞分化的影響:ALK5/Smad2訊息傳遞路徑的角色 | zh_TW |
| dc.title | Effect of TGF-β1 on the Differentiation of Human Apical Papilla Cells: Role of ALK5/Smad2 Signaling | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃富美,張美姬(Mei-Chi Chang) | |
| dc.subject.keyword | 轉化生長因子β1,人類牙根尖細胞,分化相關標誌物,抑制劑,經典訊號傳遞路徑, | zh_TW |
| dc.subject.keyword | TGF-β1,human apical papilla cells,differentiation related markers,SB431542,inhibitor,ALK5/Smad2 signaling pathway, | en |
| dc.relation.page | 113 | |
| dc.identifier.doi | 10.6342/NTU201802005 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-07-30 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 臨床牙醫學研究所 | zh_TW |
| 顯示於系所單位： | 臨床牙醫學研究所 | |
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