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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭景暉(Jiiang-Huei Jeng) | |
dc.contributor.author | Lin-I Emily Chen | en |
dc.contributor.author | 陳令儀 | zh_TW |
dc.date.accessioned | 2021-06-12T18:08:58Z | - |
dc.date.available | 2008-02-19 | |
dc.date.copyright | 2008-02-19 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-11-29 | |
dc.identifier.citation | 1. Al-Hiyasat AS, Darmani H, Milhem MM. Cytotoxicity evaluation of dental resin composites and their flowable derivatives. Clin Oral Investig 2005;9:21-5.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27540 | - |
dc.description.abstract | 許多研究發現,複合樹脂能釋放具有高度細胞毒性的單體bis-GMA到口腔中,這可能對人體造成局部、或全身性的不良影響。牙科材料的單體可能會引起過量的活性氧物質產生,進而造成細胞週期的錯亂及遏止。因為細胞週期是由許多細胞週期調節者緊密調控,因此此實驗將經由探討bis-GMA對人類牙髓細胞的細胞週期調節者Cdc2、Cdc25C、Myt1、p21、p53、p-Cdc2、p-Cdc25C及Bad的影響,進而了解bis-GMA如何影響細胞週期。另一方面,基質金屬蛋白酶是調控人類牙髓中細胞外間質的維持最重要的蛋白酶。本實驗將探討bis-GMA對於牙髓細胞中基質金屬蛋白酶-2、-9、以及基質金屬蛋白酶組織抑制因子-2的影響。因鹼性磷酸酶為牙本質形成過程中分化能力的指標,本實驗也將探討bis-GMA對於牙髓細胞中鹼性磷酸酶表現的影響。
實驗步驟首先將0到0.25mM的bis-GMA加入人類牙髓細胞作用24小時。接著觀察細胞型態的改變,並由牙髓細胞萃取tRNA進行反轉錄聚合酶鏈鎖反應,或抽出蛋白進行凝膠電泳和蛋白質轉漬。細胞培養液則以酶譜法來驗明由牙髓細胞釋放的基質金屬蛋白酶-2及基質金屬蛋白酶-9。另一組牙髓細胞則培養於0到0.075mM的bis-GMA內5天,再進行MTT細胞毒性偵測以及鹼性磷酸酶染色。 實驗結果顯示bis-GMA能夠誘發人類牙髓細胞型態的改變。典型人類牙髓細胞為細長紡錘形並具有延伸的細胞突起。在暴露於bis-GMA後,人類牙髓細胞呈圓形或殼狀。細胞在高濃度的bis-GMA會因失去活性而和培養盤底部脫離。Bis-GMA會抑制牙髓細胞Cdc2和Cdc25C蛋白及促進Myt1、p21和p53蛋白的產生,並加強p21 mRNA及抑制Cdc2、cyclin B1、和Cdc25C mRNA的表現。這些結果指出bis-GMA能經由和細胞週期有關的蛋白的異常調控進而遏止牙髓細胞的細胞週期。 Bis-GMA能增強牙髓細胞內基質金屬蛋白酶-2及基質金屬蛋白酶-9的蛋白和mRNA的表現。牙髓細胞基質金屬蛋白酶-2的表現比基質金屬蛋白酶-9表現要強。由牙髓細胞的培養液發現,牙髓細胞能產生較強的前基質金屬蛋白酶-2明膠酵素活動,及較弱的前基質金屬蛋白酶-9明膠酵素活動。Bis-GMA能顯著減少前基質金屬蛋白酶-2,但對前基質金屬蛋白酶-9則沒有顯著的影響。相反地,bis-GMA會誘發基質蛋白酵素抑制劑-2蛋白的表現。所以總括來說,bis-GMA對基質金屬蛋白酶-2、基質金屬蛋白酶-9的mRNA及蛋白表現、和對基質蛋白酵素抑制劑-2的蛋白的誘發以及對釋放出的前基質金屬蛋白酶-2的抑制,會造成過剩的基質金屬蛋白酶活動,進而加速細胞外間質的破壞。 本研究也發現低濃度的bis-GMA並不會對牙髓細胞產生顯著的細胞毒性,但是會降低鹼性磷酸酶的活性。這個結果顯示低濃度的bis-GMA會抑制牙髓細胞在牙本質形成過程中所需的分化能力,進而阻礙牙本質的形成。較高濃度的bis-GMA則會對牙髓細胞造成顯著的細胞毒性。 總結來說bis-GMA會造成牙髓細胞細胞型態和細胞週期的異常、不均衡的基質金屬蛋白酶活動、及干擾牙髓細胞的分化能力。已有研究指出複合樹脂在較深及牙髓已暴露的窩洞和牙髓的毀壞比銀粉更有關聯性,但是bis-GMA對人類牙髓組織的影響仍需更多研究來證實。 | zh_TW |
dc.description.abstract | In previous studies, it was found that the highly cytotoxic dental monomer bis-GMA could be released from resin-based dental materials into the oral environment, which could potentiate both systemic and local adverse effects. Dental monomers could induce excessive production of reactive oxygen species (ROS), leading to cell cycle aberrations and cell cycle arrest. Since cell cycles are tightly regulated by various cell cycle regulators, the effect of bis-GMA to the expression of the cell cycle regulators Cdc2, Cdc25C, Myt1, p21, p53, p-Cdc2, p-Cdc25C, and Bad was investigated in order to understand the mechanism of bis-GMA-induced cell cycle aberrations in dental pulp cells. On the other hand, the matrix metalloproteinases (MMPs) are the major proteinases regulating the maintenance of extracellular matrix (ECM) of human dental pulp. The effect of bis-GMA on the expression of MMP-2, MMP-9, and tissue inhibitors of metalloproteinases (TIMP)-2 in human dental pulp cells were investigated. Dental pulp cells were also investigated for their expression of alkaline phosphatase, a differentiative marker for dentinogenesis after treatment of bis-GMA.
Human dental pulp cells were treated with increasing concentration of bis-GMA (0 – 0.25mM) for 24 hours, before morphological changes were observed. Total RNA was isolated from these pulp cells for reverse transcription-polymerase chain reaction (RT-PCR). Proteins were also extracted from these pulp cells to run for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. Medium from the dental pulp cells were collected for zymography in identification of MMP-2 and MMP-9 released by pulp cells into the conditioned medium. Another group of dental pulp cells were cultured in bis-GMA (0 – 0.075mM) for 5 days for MTT cytotoxicity test and alkaline phosphatase (ALP) staining. Bis-GMA induced morphological changes in human pulp cells, which characteristically exhibited a long spindle shape with extended cellular processes, to a retracted, rounded, and shell-like appearance. Some cells were detached from culture well indicating loss of vitality at higher concentrations. Bis-GMA also inhibited Cdc2 and Cdc25C production, with promotion in protein production of Myt1, p21, and p53. Bis-GMA treatment induced the mRNA expression of p21 with inhibition in Cdc2, cyclin B1, and Cdc25C mRNA expression. These suggested that bis-GMA could induce cell cycle arrest by aberrant regulation of cell cycle-related proteins in dental pulp cells. Bis-GMA was found to induce both the expression of MMP-2 and MMP-9 mRNA and protein level in dental pulp cells, with generalized weaker expression for MMP-9 compared to MMP-2. A stronger gelatinolytic activity of pro-MMP-2 and a weaker activity of pro-MMP-9 were also found in the conditioned medium of dental pulp cells, whereas little active forms of MMP-2 or MMP-9 were detected. Bis-GMA was found to significantly decrease the release of pro-MMP2 in the conditioned media, with no apparent effect on the release of pro-MMP-9. In contrast to MMPs, the inhibitor of MMPs, TIMP-2 protein level was induced by bis-GMA treatment. Therefore, the combined effects of bis-GMA in induction of MMP-2 and MMP-9 mRNA and protein level, the induction in TIMP-2 and reduction in the release of the latent form of MMP-2 could lead to a net excess in MMPs activities promoting accelerated ECM degradation. Our study also found that low concentrations of bis-GMA which showed no significant cytotoxicity on dental pulp cells demonstrated reduced ALP activities. This result suggested that low concentrations of bis-GMA had an inhibitory effect on the differentiative ability of pulp cells for dentinogenesis, which could lead to interferences in dentine formation. At higher concentrations, bis-GMA exhibited a significant cytotoxic effect on dental pulp cells. In conclusion, bis-GMA could lead to aberrations in cellular morphology and cell cycle regulation, imbalanced MMPs activities, and disturbances in differentiative activities of dental pulp cells. In previous studies, dentin bonding agents have been found to cause inflammatory changes, dilation and congestion of blood vessels, production of irregular dentine, and odontoblastic displacement. Composite restorations were also found to be associated with more pulpal breakdown in deep and pulpally exposed cavities in comparison to amalgam restorations. However, further studies in vitro and in vivo were required to investigate effect of bis-GMA to human pulp tissues. | en |
dc.description.provenance | Made available in DSpace on 2021-06-12T18:08:58Z (GMT). No. of bitstreams: 1 ntu-96-R94422009-1.pdf: 1514337 bytes, checksum: 636c069a3850477ae9183d9b436c5537 (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 口試委員會審定書………………………………………………………………………i
誌謝 (Acknowledgement)……………………………………….……………………...ii 中文摘要 (Chinese Abstract)…………………………………………………………..iv 英文摘要 (English Abstract)…………….…………………………………………….vii 目錄 (Table of Content)…………………………………………………………………x 表目錄 (Table of Content for Tables)………………………………………………...xiii 圖目錄 (Table of Content for Figures)………………………………………………..xiv 第一章 (Chapter One) Introduction…….………………………………………….…...1 1.1 Historical Overview……………………………………………………………1 1.2 Release of Monomers…………………………………………………………..2 1.3 Monomer Cytotoxicity…………………………………………………………5 1.4 Apoptosis……………………………………………………………...………..6 1.5 Genotoxic Effect……………………………………………………...………..8 1.6 Reactive Oxygen Species (ROS) and Glutathione (GSH)…………...……….10 1.7 Cell Cycle Overview………………………………………………………….12 1.7.1 Cyclin B…………………………………………………………..16 1.7.2 CDK1 – Cdc2…………………………………………………….17 1.7.3 Cdc25C……………………………………………………….…..18 1.7.4 The p53 Tumor Suppressor Gene………………………………...19 1.7.5 p21………………………………………………………………..20 1.7.6 Bad………………………………………………………………..21 1.8 Matrix Metalloproteinases (MMP)……………………………………………22 1.8.1 MMP Structure…………………………………………………...24 1.8.2 Gelatinase (MMP-2 and MMP-9)………………………………..25 1.8.3 Tissue Inhibitors of Metalloproteinases (TIMP)…………………26 1.9 Collagen Secretion and Alkaline Phosphatase (ALP) Activities…………..…28 第二章 (Chapter Two) Materials and Methods..……………………………………...30 2.1 Materials……………………………………………………………………..30 2.2 Cell Culture………………….……………………………………………….30 2.3 Effect of Bis-GMA on Human Dental Pulp Cells…………………………….31 2.4 Reverse Transcription Polymerase Chain Reaction (RT-PCR)………………..32 2.4.1 Isolation of total RNA…………………………………………………32 2.4.2 RNA Quantitation……………………………………………………...33 2.4.3 Reverse Transcription (RT)……………………………………………34 2.4.4 Polymerase Chain Reaction (PCR)…………………………………….35 2.5 Western Blot Analysis…………………………………………………………38 2.5.1 Sample Preparation…………………………………………………….38 2.5.2 Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis (SDS-PAGE)………………………………………………………….39 2.5.3 Western Blot…………………………………………………………...40 2.6 Zymography…………………………………………………………………...42 2.7 MTT…………………………………………………………………………...43 2.8 Alkaline Phosphatase (ALP) Stain…………………………………………….44 2.9 Collagen Content Assay……………………………………………………….45 2.9 Statistical Analysis…………………………………………………………….47 第三章 (Chapter Three) Results……………………………………………………….48 3.1 Morphological Alterations of Human Pulp Cells Induced by Bis-GMA……..48 3.2 Effects of Bis-GMA to Cell-Cycle Related mRNA Expression of Human Pulp Cells….……………………………………………………………………….49 3.3 Effects of Bis-GMA to the Level of Cell Cycle Regulatory Proteins…...…….50 3.4 Effects of Bis-GMA to Expression of MMPs and TIMPs…………………….52 3.5 Effects of Bis-GMA to MMPs Released into Cultural Medium………………52 3.6 Cytotoxic Effects of Bis-GMA to the Vitality of Dental Pulp Cells…………..53 3.7 Effects of Bis-GMA to Alkaline Phosphatase (ALP) Activities and Collagen Content…………………………………………………………..……………53 第四章 (Chapter Four) Discussion……...…………..…………………………………55 4.1 Morphological Aberrations…………………………………………….……...55 4.2 Cell Cycle Aberrations…………………………………………….…………..56 4.3 Effects of Bis-GMA on the Expression of MMPs and TIMPs………………..66 4.4 Inhibition in Collagen Content and Alkaline Phosphatase (ALP) Activities.…74 4.5 Conclusion…………………………………………………………………….75 參考文獻 (References)………..…………………………………………….…………76 | |
dc.language.iso | en | |
dc.title | Bis-GMA對人類牙髓細胞細胞週期調節、基質金屬蛋白酶表現、及鹼性磷酸酶活性之影響 | zh_TW |
dc.title | The Effect of Bis-GMA on Cell Cycle Regulation, Expression of Matrix Metalloproteinases, and Alkaline Phosphatase Activities in Human Dental Pulp Cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-1 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 陳敏慧(Min-Huey Chen) | |
dc.contributor.oralexamcommittee | 林哲堂 | |
dc.subject.keyword | 基質金屬蛋白酶,細胞週期,鹼性磷酸酶, | zh_TW |
dc.subject.keyword | Bis-GMA,cell cycle,matrix metalloproteinases,alkaline phosphatase, | en |
dc.relation.page | 109 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-11-30 | |
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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