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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭景暉 | |
dc.contributor.author | Yu-An Huang | en |
dc.contributor.author | 黃郁安 | zh_TW |
dc.date.accessioned | 2021-05-14T17:42:08Z | - |
dc.date.available | 2018-09-24 | |
dc.date.available | 2021-05-14T17:42:08Z | - |
dc.date.copyright | 2015-09-24 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-08-19 | |
dc.identifier.citation | Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). The extracellular matrix of animals.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/4417 | - |
dc.description.abstract | 實驗目的:轉化生長因子-β1 (Transforming growth factor-β1,TGF-β1) 對於牙髓的修復與牙本質生成扮演重要的角色,包含細胞外基質 (Extracellular matrix,ECM) 的重塑。纖維蛋白溶解活化系統 (plasminogen activation system,PA system) 參與ECM的調控,然而關於TGF-β1和PA系統的相關性仍尚未瞭解。本研究目的在探討TGF-β1對於人類牙髓細胞的生長以及PA系統的影響,還有相關訊息路徑傳導。
實驗方法:使用0.5, 1, 5, 10, 25 ng/mL TGF-β1刺激人類牙髓細胞,以細胞存活率分析 (MTT assay)、反轉錄聚合酶連鎖反應 (RT-PCR)、西方墨點法 (Western Blot) 及免疫螢光法 (Immunofluorescence),在24小時和5天兩個時間點檢測TGF-β1在細胞生長和尿激酶型纖維蛋白溶解酶原活化因子 (urokinase-type plasminogen activator, uPA)、尿激酶型纖維蛋白溶解酶原活化因子受體 (urokinase-type plasminogen activator receptor, uPAR)、纖維蛋白溶解酶原活化因子抑制劑第一型 (plasminogen activator inhibitor-1, PAI-1)、第一型膠原蛋白 (collagen I) 的影響。同時也分析TGF-β1對人類牙髓細胞Smad2/3、TAK1 以及ERK1/2的活化;在某些實驗組別,則加入SB431542 (ALK5/Smad2/3抑制劑)、5z-7-oxozeaenol (TAK1抑制劑)、U0126 (MEK/ERK抑制劑),探討可能參與調控細胞反應的訊息傳遞路徑。 實驗結果:TGF-β1有抑制牙髓細胞生長的趨勢,且SB431542可以逆轉這個趨勢,U0126則無法逆轉,而5z-7-oxozeaenol的加入使得細胞數目下降更多。TGF-β1可以促進PAI-1和uPAR基因和蛋白的表現,uPA的基因和蛋白表現則被抑制,collagen I沒有顯著改變。SB431542、5z-7-oxozeaenol和U0126可以影響TGF-β1對PAI-1/uPAR的表現,uPA 則會受到SB431542和5z-7-oxozeaenol影響。此外,TGF-β1能活化p-Smad2、p-Smad3、p-TAK1和p-ERK等訊息活化。 結論:TGF-β1對於牙髓細胞的訊息路徑調控是相當複雜的,透過Smad (ALK5/Smad2/3) 和Non-Smad (TAK1 及 MEK/ERK) 路徑,TGF-β1可以調控PAI-1/uPA/uPAR,影響ECM的堆積與重塑。本實驗結果對於牙髓細胞的修復有進一步瞭解,期望在未來再生醫學方面的研究能有所幫助。 | zh_TW |
dc.description.abstract | Aim: Transforming growth factor-β1 (TGF-β1) plays a role in the repair and dentinogenesis of human dental pulp, including the deposition and remodeling of extracellular matrix (ECM). Plasminogen activation system regulates ECM degradation. However the relationship between TGF-β1 and plasminogen activation system is not fully clear. The purpose of this study was to investigate the effect of TGF-β1 on plasminogen activation system of human dental pulp cells and its related signaling pathways.
Materials and methods: Primary human dental pulp cells were treated with 0.5, 1, 5, 10, 25 ng/mL of TGF-β1. MTT assay, Reverse Transcription Polymerase Chain Reaction) (RT-PCR), Western Blot and Immunofluorescence were used to detect the effect of TGF-β1 on cell viability and expression of urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) and collagen I at 24 hrs and 5 days. The activation of Smad2, TAK1, and ERK was also addressed. Besides, cells were pretreated with SB431542 (an ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (a TAK1 inhibitor), U0126 (a MEK/ERK inhibitor) for examining the related signaling pathways. Results: TGF-β1 slightly inhibited cell proliferation in a dose-dependent manner, which could be reversed by SB431542 but not be reversed by U0126, and the cell number decreased more when adding 5z-7-oxozeaenol. TGF-β1 up-regulated PAI-1 and uPAR expression, whereas uPA was down-regulated and the changes of collagen I was not obvious. SB431542, 5z-7-oxozeaenol and U0126 generally could reverse the expression of TGF-β1 on PAI-1/uPAR; besides, SB431542 and 5z-7-oxozeaenol might have influence on uPA expression. Furthermore, TGF-β1 induced the activation of p-Smad2, p-Smad3, p-TAK1, and p-ERK signaling. Conclusion: The regulation of signaling pathway on TGF-β1-treated dental pulp cells is complicated. TGF-β1 can affect PAI-1/uPA/uPAR expression through Smad/ALK5 and Non-Smad (TAK1 and MEK/ERK) pathways and therefore affect the deposition and remodeling of ECM. These results give an aspect to understand the mechanism of pulpal repair, and can be helpful for further investigation of pulpo-dentin regeneration. | en |
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dc.description.tableofcontents | 口試委員會審定書..........................................i
誌謝...................................................ii 中文摘要...............................................iii ABSTRACT................................................v 目錄..................................................vii 縮寫表.................................................ix 第一章 導論............................................1 1.1 牙髓-牙本質複合體 (dentin-pulp complex)..............1 1.1.1 牙髓的修復機制...................................1 1.1.2 生長轉化因子-β (TGF-β) 與牙髓-牙本質複合體.........2 1.2 轉化生長因子-β (transforming growth factor-β).......3 1.2.1 TGF-β異型體與胜肽結構.............................3 1.2.2 TGF-β 受體......................................4 1.2.3 TGF-β 訊息傳遞...................................4 1.3 細胞外基質 (extracellular matrix)...................5 1.4 纖維蛋白溶解活化系統 (plasminogen activation system,PA system)..............................................6 第二章 實驗目的與虛無假設 ................................8 第三章 材料與方法.......................................9 3.1 材料...............................................9 3.2 培養牙髓細胞.......................................10 3.3 藥物對牙髓細胞形態影響 (cell morphology)............10 3.4 細胞存活率分析 (MTT assay) (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)................11 3.5 反轉錄聚合酶連鎖反應 (reverse transcription polymerase chain reaction) (RT-PCR)...............................11 3.5.1 細胞的RNA萃取...................................12 3.5.2 RNA定量........................................13 3.5.3 反轉錄 (reverse transcription,RT)..............13 3.5.4 聚合酶連鎖反應 (polymerase chain reaction,PCR)..14 3.5.5 PCR產物電泳分析.................................14 3.6 西方墨點法 (western blot)..........................15 3.6.1 細胞的蛋白質萃取.................................15 3.6.2 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-PAGE)..15 3.6.3 蛋白質樣本的轉印.................................17 3.6.4 免疫墨點法 (immune-blot)........................17 3.7 免疫螢光法 (immunofluorescence)....................18 3.8 統計..............................................19 第四章 結果...........................................20 4.1 細胞形態觀察.......................................20 4.2 TGF-β1對細胞存活率的影響- MTT assay.................20 4.3 TGF-β1對PAI-1/ uPA/ uPAR/ collagen I的影響,以及抑制劑的調控 - RT-PCR & western blot.........................21 4.3.1 TGF-β1處理5天& 24小時...........................21 4.3.2 加入SB421542、5z-7-oxozeaenol與U0126處理5天&24小時.....................................................21 4.4 TGF-β1活化訊息路徑 - western blot & immunofluorescence.....................................22 第五章 討論...........................................23 5.1 TGF-β1對於細胞存活率的影響..........................23 5.2 TGF-β1對PAI-1/ uPA/ uPAR 的影響....................24 5.3 TGF-β1對collagen I的影響...........................26 5.4 TGF-β1對Smad/non-Smad訊息路徑的影響.................26 第六章 結論...........................................28 參考文獻................................................29 附錄...................................................39 | |
dc.language.iso | zh-TW | |
dc.title | 轉化生長因子-β1對人類牙髓細胞之纖維蛋白溶解活化系統的調控與訊息路徑傳導 | zh_TW |
dc.title | Regulation and Signaling of TGF-β1 on the plasminogen activation system of human dental pulp cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 呂炫?,林俊彬,黃何雄,張美姬 | |
dc.subject.keyword | 人類牙髓細胞,轉化生長因子-β1,尿激?型纖維蛋白溶解?原活化因子,尿激?型纖維蛋白溶解?原活化因子受體,纖維蛋白溶解?原活化因子抑制劑第一型, | zh_TW |
dc.subject.keyword | human dental pulp cell,TGF-β1,PAI-1,uPA,uPAR, | en |
dc.relation.page | 57 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2015-08-19 | |
dc.contributor.author-college | 牙醫專業學院 | 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 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-104-1.pdf | 3.29 MB | Adobe PDF | 檢視/開啟 |
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