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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 | Wei-Ling Huang | en |
dc.contributor.author | 黃韋綾 | zh_TW |
dc.date.accessioned | 2021-06-16T17:50:59Z | - |
dc.date.available | 2014-09-17 | |
dc.date.copyright | 2012-09-17 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64501 | - |
dc.description.abstract | 實驗目的:轉型生長因子1 (Transforming growth factor-beta1 ,TGF-
β1)對於牙髓組織的修復與牙本質生成扮演重要的角色。本實驗的目 的在於探討TGF-β1對牙髓細胞生長分化所造成影響時TAK1 以及p38所扮演的 角色。 實驗方法:先加入SB203580 (p38抑制劑),5Z-7-oxozeaenol (TAK1抑制劑)做前處理30分鐘,再使用TGF-β1對人類牙髓細胞做刺激。以MTT 測定,反轉錄鏈聚合反應(RT-PCR),鹼性磷酸酶染色 (ALP staining),膠原蛋白定量測定(Sircol Collagen Assay) 來觀察我們的發現。 實驗結果: TGF-β1 (10 ng/ml)並不會誘發牙髓細胞增生,SB203580 (p38抑制劑)亦不會改變TGF-β1 (10 ng/ml)在牙髓細胞的影響。然而5Z-7-oxozeaenol(TAK1抑制劑)卻會降低細胞的存活能力。TGF-β1 (0.1 ng/ml) 些微促進人類牙髓細胞的鹼性磷酸酶的活性會被SB203580 與5Z-7-oxozeaenol所逆轉,而TGF-β1在較高濃度時 (10 ng/ml)會抑制人類牙髓細胞的鹼性磷酸酶的活性而抑制現象無法被SB203580 與5Z-7-oxozeaenol逆轉。透過SB203580 與5Z-7-oxozeaenol,TGF-β1 (5 ng/ml)促進牙髓細胞中膠原蛋白的合成以及TGF-β1 (0.1 ng/ml) 促進鹼性磷酸酶的活性,會逆轉其影響。。然而反轉錄鏈聚合反應實驗亦證實SB203580 與5Z-7-oxozeaenol會抑制牙髓細胞原本因TGF-β1 (10 ng/ml) 促進collagen的基因 表現,但對ALP基因降低表現並無法改變。 結論: TAK1似乎對誘導牙髓細胞的生長能力有所影響。透過TAK1-p38的訊息傳導途徑,TGF-β1 (0.1 ng/ml) 可促進人類牙髓細胞的鹼性磷酸酶的活性以及TGF-β1 (5 ng/ml)可促進牙髓細胞中膠原蛋白的合成。TGF-β1 在高濃度時(10 ng/ml) 無法透過TAK1 - p38路徑抑制人類牙髓細胞的鹼性磷酸酶的活性。 | zh_TW |
dc.description.abstract | Aim: Transforming growth factor β1 (TGF-β1) plays a role in repair and dentinogenesis in dental pulp cells. The purpose of this study is to investigate whether TGF-β1 stimulates the signaling pathways, TAK1 and p38, thereby influence the morphological changes, cell proliferation, collagen turnover and alkaline phosphatase
in human dental pulp cells in vitro. Materials and Methods: Primary-cultured human dental pulp cells were pre-treated with SB203580 (p38 inhibitor) or 5Z-7-oxozeaenol(TAK1 inhibitor) 30 minutes before adding TGF-β1. Morphology of pulp cells was observed under light microscopy (100X). Cell proliferation was evaluated by MTT assay. Cell differentiation and mineralization were evaluated by alkaline phosphatase (ALP) staining and quantitative assay. Changes in mRNA expression (ALP and collagen) were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Collagen content was determined by Sircol Collagen assay. Result : In the dental pulp cells, TGF-β1 (10ng/ml) showed little effect on cell number and cell size under light microscopy (100X) and MTT assay. 5Z-7-oxozeaenol(TAK1 inhibitor) showed decrease in the number of dental pulp cells markedly but SB203580 (p38 inhibitor) did not. TGF-β1 (10ng/ml) decreased the ALP activity but SB203580 and 5Z-7-oxozeaenol could not reverse the effect. TGF-β1 (0.1 ng/ml) mildly increased the ALP expression and TGF-β1 (5 ng/ml) increased the collagen formation. These effects could be reversed by SB203580 and 5Z-7-oxozeaenol. According to the RT-PCR, SB203580 and 5Z-7-oxozeaenol could reverse the up-regulatory of collagen expression by TGF-β1 (10ng/ml). However, down-regulatory of ALP gene expression caused by TGF-β1 (10ng/ml) could not be reversed by SB203580 and 5Z-7-oxozeaenol. Conclusion: TAK1 may increase proliferation of human dental pulp cells. TGF-β1 (0.1 ng/ml) can increase ALP activity and TGF-β1 (5 or 10 ng/ml) can induce collagen formation in dental pulp cells via TAK1-p38 pathway. TAK1-p38 pathway may have no influence in down-regulatory of ALP activity in TGF-β1 (10 ng/ml). | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T17:50:59Z (GMT). No. of bitstreams: 1 ntu-101-R98422020-1.pdf: 2035212 bytes, checksum: f7a9c047e8a04851749110d3536dc3ba (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | Table of Contents
中文摘要 ...................................................................................i Abstract .....................................................................................ii Chapter I Introduction…………………………….................1 1.1 Reparative mechanism of pulpal-dentin complex ..................... 1 1.2 Tissue engineering to regeneration of pulp and dentin ………………………………………………………………...4 1.3 Transforming Growth Factor–beta ……………........................ 5 1.3.1 Introduction of TGF-β............................................,......... 5 1.3.2 TGF-β isoforms and their peptide structure .................... 5 1.3.3 TGF-β in dental pulp ....................................................... 6 1.4 Signal transduction pathways of TGF-β1................................ 7 1.4.1 Mitogen activated protein kinase (MAPK) pathway...... 7 1.4.2 TAK1 and TGF-β1-signaling..........................................8 1.4.3 p38 MAPK signaling pathway........................................ 8 1.4.4 p38 in dental pulp........................................................... 9 1.5 Alkaline phosphatase and its role in tissue mineralization ..... 10 1.6 The purpose of this study ……………………………………11 Chapter II Materials & Methods ..........................................12 3.1 Materials ................................................................................. 12 3.2 Culture of human dental pulp cells.......................................... 12 3.3 Morphology of human dental pulp cells ................................. 13 3.4 MTT assay .............................................................................. 13 3.5 Alkaline phosphatase (ALP) staining ..................................... 14 3.6 Quantitative assay of ALP activity.......................................... 15 3.6.1 cell lysate collection ...................................................... 15 3.6.2 ALP activity measurement.............................................. 16 3.7 Collagen Content Assay ......................................................... 16 3.8 Reverse Transcription Polymerse Chain Reaction (RT-PCR). 18 3.8.1 Isolation of total RNA ................................................... 18 3.8.2 RNA Quantitation........................................................... 19 3.8.3 Reverse transcription (RT) ..........................................20 3.8.4 Polymerase Chain Reaction (PCR)................................ 20 3.9 Statistical analysis.................................................................... 21 Chapter III Results .................................................................22 4.1 Morphological observation on human dental pulp cell .......... 22 4.2 Effect of TGF-β1 on cell viability of human dental pulp cells and its modulation by SB203580 and 5Z-7-osozeaenol --- MTT assay…………………............................................................ 23 4.3 Effect of TGF-β1 on type I collagen formation of dental pulp cell and its modulation by SB203580 and 5Z-7-osozeaenol --- Sircol assay………………………………….......................... 24 4.4 Effect of TGF-β1 on ALP activity of pulp cells and its modulation by SB203580 and 5Z-7-osozeaenol --- ALP staining and ALP quantitative assay…….............................................. 25 4.5 Effect of SB203580 and 5Z-7-oxozeaenol on the TGF-β1-induced alterations of ALP and collagen expression in dental pulp cells ...................................................................... 27 Chpater IV Discussion ........................................................29 5.1 Effect of p38 and TAK1 on cell viability and proliferation of human dental pulp cells……………………………….…….29 5.2 Effect of p38 and TAK1 on matrix turnover of human dental pulp cells…………………………………………………….30 5.3 Effect of p38 and TAK1 on cell differentiation of human dental pulp cells…………………………………………......32 Chapter V Conclusion …........................................................35 References .....................................................................36 Tables Table 1: Cells and extracellular matrix components found in dentin and pulp ......................................................................................................... 45 Table 2: Protocol for Sircol Collagen Assay standard sample preparation............................................................................................... 46 Table 3: PCR Primers Sense Sequences, Antisense Sequence, Base Pairs......................................................................................................... 47 Table 4: MTT assay for TGF-β1 and SB203580................................... 48 Table 5: MTT assay for TGF-β1 and 5Z-7-oxozeaenol.......................... 49 Figures Figure 1: Primary, secondary and tertiary dentin …………………....... 50 Figure 2: Reparative & Reactionary dentinogenesis……………….... 50 Figure 3: Tissue engineering in endodontic field………….....................51 Figure 4: Signal Transduction Pathway of TGF-s superfamily................52 Figure 5: MAPK Pathway........................................................................ 53 Figure 6: The TβRI–TRAF6 interaction is required for activation of the TAK1 –p38 / JNK pathway,..................................................................... 54 Figure 7: Protocol for stock substrate solution in Alkaline Phosphatase (ALP) Stain ………………………………………………………...…..55 Figure 8: Protocol for preparation of PBS with Ca2+, Mg2+ for ALP Stain......................................................................................................... 56 Figure 9: Preparation of incubation solution for ALP staining ……………………………………………………………...…57 Figure 10: Protocol for total RNA isolation using invitrogen SuperScriptTM III First Strand Synthesis System..................................... 58 Figure 11: Morphology of human dental pulp cells after pre-treat SB203580 30min then treatment with TGF-β1...................................... 60 Figure 12: Morphology of human dental pulp cells after pre-treat 5Z-7-oxozeaenol 30min then treatment with TGF-β1............................ 62 Figure 13: MTT assay: 5 days incubation of pulp cells under the treatment of TGF-β1 (10 ng/ml) & SB203580(10 or 20 μM)……......... 63 Figure 14: MTT assay: 5 days incubation of pulp cells under the treatment of TGF-β1 (10 ng/ml) & 5Z-7-oxozeaenol(1 or 5 μM)……... 64 Figure 15-1: Sircol collagen assay: 5 days incubation of pulp cells under the treatment of TGF-β1 (5 ng/ml) & SB203580(10 or 20 μM)............. 65 Figure 15-2: Sircol collagen assay: 5 days incubation of pulp cells under the treatment of TGF-β1 (5 ng/ml) & 5Z-7-oxozeaenol(1 or 5 μM)....... 66 Figure 16: ALP staining: TGF-β1 (5 or 10 ng/ml) down-regulates the ALP activity of dental pulp cells after 5 days incubation. However, TGF-β1 (0.1 ng/ml) up-regulates the ALP activity…………………....................67 Figure 17: (a)ALP staining and (b)ALP quantitative assay of pulp cells were pretreated with SB203580 (10 or 20 μM) for 30 min before addition of TGF-β1(0.1 ng/ml) and then co-incubated for 5 days..…………... . . 69 Figure 18: (a)ALP staining and (b)ALP quantitative assay of pulp cells were pretreated with SB203580 (10 or 20 μM) for 30 min before addition of TGF-β1(10 ng/ml) and then co-incubated for 5 days.......................... 71 Figure 19: ALP staining of pulp cells was pretreated with 5Z-7-oxozeaenol (1 or 5 μM) for 30 min before addition of TGF-β1 (0.1ng/ml) and then co-incubated for 5 days.……………………….......72 Figure 20: (a)ALP staining and (b)ALP quantitative assay of pulp cells was pretreated with 5Z-7-oxozeaenol (1 or 5 μM) for 30 min before addition of TGF-β1(10 ng/ml) and then co-incubated for 5 days............ 74 Figure 21: RT-PCR: 5Z-7-oxozeaenol and SB203580 could not reverse the down-regulatory of ALP gene expression caused by TGF-β1 (10 ng/ml)................................................................................................. 75 Figure 22: RT-PCR: SB203580 could reverse the up-regulatory of collagen type I gene expression caused by TGF-β1 (10ng/ml)……........76 | |
dc.language.iso | en | |
dc.title | TGF-beta對人類牙髓細胞的影響:p38與TAK1之角色 | zh_TW |
dc.title | Role of p38 and TAK1 on TGF-β-induced effect on human dental pulp cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林俊彬,李勝揚,涂明君,張美姬 | |
dc.subject.keyword | 牙髓細胞,轉型生長因子,鹼性磷酸酶,p38,TAK1, | zh_TW |
dc.subject.keyword | dental pulp cells,TGF-β,alkaline phosphoatase,p38,TAK1, | en |
dc.relation.page | 76 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-14 | |
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 |
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