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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 鄭景暉 | |
dc.contributor.author | Yi-Chieh Chen | en |
dc.contributor.author | 陳頤潔 | zh_TW |
dc.date.accessioned | 2021-06-08T03:28:40Z | - |
dc.date.copyright | 2019-08-26 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-17 | |
dc.identifier.citation | Cao Z, Zhang H, Zhou X, Han X, Ren Y, Gao T, Xiao Y, de Crombrugghe B, Somerman MJ & Feng JQ (2012) Genetic evidence for the vital function of osterix in cementogenesis. Journal of Bone and Mineral Research 27: 1080-1092.
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Xu L, Meng F, Ni M, Lee Y & Li G (2013) N-cadherin regulates osteogenesis and migration of bone marrow-derived mesenchymal stem cells. Molecular biology reports 40: 2533-2539. Yang B, Chen G, Li J, et al. (2012) Tooth root regeneration using dental follicle cell sheets in combination with a dentin matrix - based scaffold. Biomaterials 33: 2449-2461. Yang G, Li X, Yuan G, Liu P & Fan M (2014) The Effects of Osterix on the Proliferation and Odontoblastic Differentiation of Human Dental Papilla Cells. Zhang F, Song J, Zhang H, et al. (2016) Wnt and BMP Signaling Crosstalk in Regulating Dental Stem Cells: Implications in Dental Tissue Engineering. Genes & diseases 3: 263-276. Zhou X, Zhang Z, Feng JQ, Dusevich VM, Sinha K, Zhang H, Darnay BG & 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: 12919-12924. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21205 | - |
dc.description.abstract | 實驗目的
探討如何修復受損牙齒組織,並保存和維持受影響牙齒的自然功能,是再生牙髓病學領域中的重要議題。因此,找到合適的生長因子和幹細胞是組織工程學及再生醫學的成功關鍵。來自人類牙根尖的幹細胞(SCAPs)被認為是牙齒組織再生的有效細胞來源,而骨形態蛋白-4(BMP-4)在其中扮演著重要的角色。本研究旨在通過分析相關標誌物的表現,來探討BMP-4對SCAPs的分化潛能和間質代謝的影響,標誌物包括:Osterix(Osx)、N-cadherin、鹼性磷酸(ALP)、第一型膠原蛋白(COL1A1)、尿激型纖溶原激活物(uPA)和纖溶原激活物抑制劑-1(PAI-1)等, 並探討其信號傳導途徑。 實驗方法 使用原代培養的SCAPs作為實驗細胞,並用不同濃度的BMP-4(0, 10, 25, 50, 100, 200 ng/ml)處理24小時後,通過RT-PCR和western blot檢測Osx、N-cadherin、ALP、COL1A1、uPA、PAI-1的表現。另外,也使用p-Smad1/5/8抑制劑(LDN193189)和p-Smad2/3抑制劑 (SB431542)來研究在SCAPs中BMP-4誘發的Smad信號傳導途徑。 實驗結果 加入BMP-4處理24小時後的的SCAPs細胞顯示:Osx、N-cadherin、ALP、COL1A1和PAI-1的mRNA和蛋白質表現量增加,而uPA的表現量降低。加入LDN193189和SB431542後,結果顯示BMP-4在Smad傳導路徑的誘導作用被抑制。LDN193189顯著降低了Smad1/5/8、Smad2/3 的磷酸化,而SB431542則顯著降低了Smad1/5/8的磷酸化。 結論 根據本篇研究結果顯示,SCAPs具有進行多譜系分化的潛力,包括成骨作用、牙齒再生和細胞外間質代謝。根據抑制劑的結果顯示,Smad1/5/8和Smad2/3這兩種途徑可能存在一些交互作用。而TGF-β的信號傳導途徑可能參與了BMP-4的信號傳導途徑。BMP-4藉由Smad傳導路徑有調節SCAPs的分化和細胞外間質代謝的角色。SCAPs和BMP-4的組合使用未來或許可以運用在牙科組織修復工程領域之中。 | zh_TW |
dc.description.abstract | Aim
Regenerative endodontics is a field dedicated to the repair and restoration of damaged dental tissues, in hopes of conserving and maintaining natural function of the affected tooth. Finding the suitable growth factors and stem cells are key factors that contribute to successful tissue engineering. Human stem cells from apical papilla (SCAPs) are known as potent cell sources for dental tissue regeneration, while the bone morphogenic protein-4 (BMP-4) is a growth factor that is vital in the growth and development of human teeth. This study was designed to investigate the impacts BMP-4 may have on the differentiation potential and matrix turnover SCAPs, by analyzing the expression of differentiation related markers, including osterix (Osx), N-cadherin, alkaline phosphatase (ALP), type I collagen (COL1A1), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). The signaling pathways underlying these effects were also explored. Materials and Methods Primary-cultured SCAPs were treated with different concentrations of BMP-4 (0, 10, 25, 50, 100, 200 ng/ml) for 24 hours. The expressions of Osx, N-cadherin, ALP, COL1A1, uPA, PAI-1 were evaluated through RT-PCR and western blot. In addition, pre-treatment of LDN193189 (a p-Smad1/5/8 inhibitor) and SB431542 (a p-Smad2/3 inhibitor) were used to investigate the Smad-dependent pathways induced by BMP-4 in SCAPs. Results SCAPs exposed to BMP-4 for 24 hours showed an increase in the mRNA and protein expressions of Osx, N-cadherin, ALP, COL1A1, and PAI-1, whereas the expression levels of uPA were decreased. Pre-treatment with inhibitors, LDN193189 and SB431542, showed inhibitory effects on the BMP-4-stimulated expressions. Moreover, the addition of LDN193189 significantly reduced the phosphorylation of Smad1/5/8 and Smad2/3. Similarly, when SB431542 was added, the phosphorylation level of Smad1/5/8 was diminished. Conclusion Our results demonstrate that SCAPs have the potential to undergo multi-lineage differentiation, including osteogenesis, odontogenesis, and extracellular matrix turnover. In addition, pre-treatment with LDN193189 significantly decreased the phosphorylation levels of Smad1/5/8 and Smad2/3, while SB431542 also reduced phosphorylation of Smad1/5/8, suggesting these two pathways may have some interaction or crosstalk with each other, and that the TGF-β signaling pathway may possibly play a role in the mechanism of BMP-4 signaling pathways. It is visible through our research that BMP-4 plays an important role in the differentiation and extracellular matrix turnover of SCAPs by regulation through the Smad-dependent signaling pathways. Thus, the knowledge obtained from this study on the combined use of SCAPs and BMP-4 may be a beneficial approach for future clinical applications in dental tissue repair and engineering. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:28:40Z (GMT). No. of bitstreams: 1 ntu-108-R05422022-1.pdf: 19083664 bytes, checksum: 35ba727830296acaecf3e5bd353c1421 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 口試委員審定書 ..............................................................................i
謝誌 .............................................................................................ii 中文摘要 ...................................................................................... iii Abstract ........................................................................................iv Chapter I. Literature Review .............................................................. 1 1.1 Regenerative endodontics ...........................................................................1 1.2 Human stem cells from apical papilla (SCAPs) ................................................ 2 1.3 Bone morphogenetic proteins (BMPs) ............................................................ 4 1.3.1 BMP family ...................................................................................... 4 1.3.2 BMP receptors and signaling pathways ..................................................... 4 1.3.3 BMP effects on human ......................................................................... 6 1.3.4 BMP effects on teeth ........................................................................... 7 1.3.5 BMP-4 and dental tissue engineering ....................................................... 7 1.4 Differentiation related markers ................................................................... 9 1.4.1 Osterix (Osx, Sp7) .............................................................................. 9 1.4.2 N-cadherin ....................................................................................... 10 1.4.3 Alkaline phosphatase (ALP) .................................................................. 11 1.4.4 Type I collagen (COL1A1) ................................................................... 13 1.4.5 Urokinase-type plasminogen activator (uPA) .............................................. 14 1.4.6 Plasminogen activator inhibitor-1 (PAI-1) .................................................. 15 Chapter II. The Purpose of the Study ................................................... 16 Chapter III. Materials and Methods ..................................................... 17 3.1 Materials ................................................................................................ 17 3.2 Culture of SCAPs ..................................................................................... 18 3.3 Reverse transcription polymerase chain reaction (RT-PCR) ................................ 19 3.3.1 Total RNA isolation ............................................................................ 19 3.3.2︎ RNA quantification ............................................................................. 21 3.3.3︎ Reverse transcription (RT) .................................................................... 21 3.3.4︎ Polymerase chain reaction (PCR) ........................................................... 22 3.4 Western blot ............................................................................................ 23 3.4.1 Protein extraction ............................................................................... 23 3.4.2 Protein quantification .......................................................................... 24 3.4.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ..................................................................................... 25 3.4.4 Transfer gel ...................................................................................... 25 3.4.5 Blocking and antibody hybridization ........................................................ 26 3.4.6 Chemiluminescence photography ............................................................ 26 Chapter IV. Results .......................................................................... 27 4.1 Effects of different concentrations of BMP-4 on SCAPs ..................................... 27 4.1.1 Effects of BMP-4 on the mRNA and protein expressions of Osx ........................ 27 4.1.2 Effects of BMP-4 on the mRNA and protein expressions of N-cadherin ............... 27 4.1.3 Effects of BMP-4 on the mRNA and protein expressions of ALP ....................... 27 4.1.4 Effects of BMP-4 on the mRNA and protein expressions of COL1A1 ................. 27 4.1.5 Effects of BMP-4 on the mRNA and protein expressions of uPA ........................ 28 4.1.6 Effects of BMP-4 on the mRNA and protein expressions of PAI-1 ..................... 28 4.1.7 Effects of BMP-4 on the phosphorylation of Smad1/5/8 and Smad2/3 ................. 28 4.2 Effects of BMP-4 with the addition of LDN193189 (p-Smad1/5/8 inhibitor) and SB431542 (p-Smad2/3 inhibitor) on SCAPs ............................................... 29 4.2.1 Effects of BMP-4 with the addition of LDN193189 and SB431542 on the expression of Osx .............................................................................. 29 4.2.2 Effects of BMP-4 with the addition of LDN193189 and SB431542 on the expression of N-cadherin ...................................................................... 29 4.2.3 Effects of BMP-4 with the addition of LDN193189 and SB431542 on the expression of ALP .............................................................................. 29 4.2.4 Effects of BMP-4 with the addition of LDN193189 and SB431542 on the expression of COL1A1 ........................................................................ 30 4.2.5 Effects of BMP-4 with the addition of LDN193189 and SB431542 on the expression of PAI-1 ............................................................................ 30 4.2.6 Effects of BMP-4 with the addition of LDN193189 and SB431542 on the phosphorylation of Smad1/5/8 and Smad2/3 ............................................... 30 4.2.7 Effects of BMP-4 with the addition of LDN193189 on the phosphorylation of Smad2/3 .......................................................................................31 Chapter V. Discussion ....................................................................... 31 Chapter VI. Conclusion ..................................................................... 38 References ..................................................................................... 40 Tables Table 1. PCR Primer Sequences ............................................................................. 46 Table 2. Protocol of Western Blot Protein Extraction Buffer ............................................ 46 Table 3. Protocol for SDS-PAGE ............................................................................ 47 Table 4A. Protocol for Western Blot SDS-PAGE Running Buffer ...................................... 48 Table 4B. Protocol for Western Blot SDS-PAGE Transfer Buffer ....................................... 48 Table 4C. Protocol for Western Blot Tween TBS .......................................................... 48 Table 5. Western Blot Primary Antibodies .................................................................. 49 Figures Figure 1. Effects of BMP-4 on the mRNA and protein expressions of Osx ........................... 50 Figure 2. Effects of BMP-4 on the mRNA and protein expressions of N-cadherin ................... 51 Figure 3. Effects of BMP-4 on the mRNA and protein expressions of ALP ........................... 52 Figure 4. Effects of BMP-4 on the mRNA and protein expressions of COL1A1 ..................... 53 Figure 5. Effects of BMP-4 on the mRNA and protein expressions of uPA ............................ 54 Figure 6. Effects of BMP-4 on the mRNA and protein expressions of PAI-1 .......................... 55 Figure 7. Effects of BMP-4 on the phosphorylation of Smad1/5/8 and Smad2/3 ..................... 56 Figure 8. Effects of BMP-4 with the addition of LDN193189 and SB431542 on the expression of Osx .................................................................................................. 57 Figure 9. Effects of BMP-4 with the addition of LDN193189 and SB431542 on the expression of N-cadherin .......................................................................................... 58 Figure 10. Effects of BMP-4 with the addition of LDN193189 and SB431542 on the expression of ALP .................................................................................................. 59 Figure 11. Effects of BMP-4 with the addition of LDN193189 and SB431542 on the expression of COL1A1 ............................................................................................ 60 Figure 12. Effects of BMP-4 with the addition of LDN193189 and SB431542 on the expression of PAI-1 ................................................................................................ 61 Figure 13. Effects of BMP-4 with the addition of LDN193189 and SB431542 on the phosphorylation of Smad1/5/8 .................................................................. 62 Figure 14. Effects of BMP-4 with the addition of LDN193189 on the phosphorylation of Smad2/3 ............................................................................................ 63 | |
dc.language.iso | en | |
dc.title | BMP-4 對於人類牙根尖細胞分化和間質代謝的影響:Smad1/5/8 和 Smad2/3 訊息傳導路徑的角色 | zh_TW |
dc.title | Effects of BMP-4 on the Differentiation and Matrix Turnover of Human Stem Cells from Apical Papilla: Role of Smad1/5/8 and Smad2/3 Signaling | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 張曉華 | |
dc.contributor.oralexamcommittee | 楊淑芬,洪善鈴,張美姬 | |
dc.subject.keyword | 骨形態蛋白-4,人類牙根尖的幹細胞, | zh_TW |
dc.subject.keyword | BMP-4,Human Stem Cells from Apical Papilla, | en |
dc.relation.page | 63 | |
dc.identifier.doi | 10.6342/NTU201903786 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2019-08-18 | |
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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