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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 | Meng-Lan Tsai | en |
| dc.contributor.author | 蔡孟嵐 | zh_TW |
| dc.date.accessioned | 2023-03-19T23:00:46Z | - |
| dc.date.copyright | 2022-10-03 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-07-20 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85369 | - |
| dc.description.abstract | 目的：10-(2-甲基丙烯酰氧基)磷酸單癸酯因具有良好的臨床治療材料特性，近年來被廣泛地使用在通用型牙科黏著劑中。然而，10-(2-甲基丙烯酰氧基)磷酸單癸酯對細胞可能造成的毒性和不良反應，以及發生的機制，目前尚未完全明瞭。本研究的目的在於探討10-(2-甲基丙烯酰氧基)磷酸單癸酯對人類牙髓細胞的細胞存活率、活性含氧物的生成、發炎反應以及細胞週期的影響。並透過分析抗氧化酶和細胞週期的調控因子的表現，進一步研究10-(2-甲基丙烯酰氧基)磷酸單癸酯所產生毒性的機制。 實驗方法：使用培養的人類牙髓細胞進行實驗，並加入最終濃度0、10、50、100、250和500 μM的10-(2-甲基丙烯酰氧基)磷酸單癸酯培養24小時，以MTT檢測細胞的存活率，使用流式細胞術以PI和DCF螢光染色分析細胞週期和活性含氧物的生成，以即時定量聚合酶連鎖反應(real-time quantitative PCR)、西方點墨法(western blot)和免疫化學螢光染色(immunofluorescence assay)來檢測抗氧化酶(Nrf2、p-Nrf2、HO-1)、發炎相關因子(COX-2、IL-6、IL-8)和細胞週期的調控因子(cyclin B1、cdc25C、p21、GADD45α、p-ATM)的基因和蛋白表現之變化。 實驗結果：人類牙髓細胞受到250和500 μM的10-(2-甲基丙烯酰氧基)磷酸單癸酯的影響，可以觀察到細胞存活率降低以及細胞形態發生變化。250 μM的10-(2-甲基丙烯酰氧基)磷酸單癸酯會讓人類牙髓細胞活性含氧物的生成增加，提升p-Nrf2和HO-1的表現，並使發炎相關因子COX-2、IL-6和IL-8的表現上升。另外，10-(2-甲基丙烯酰氧基)磷酸單癸酯在250 μM的時候也會造成人類牙髓細胞之細胞週期調節因子發生改變，p-ATM、p21和GADD45α的表現上升，cyclin B1和cdc25C表現下降，細胞週期出現受到干擾的現象，sub-G0/G1、G0/G1和G2/M phase的細胞比例都有增加趨勢。 結論：由本實驗結果推測，10-(2-甲基丙烯酰氧基)磷酸單癸酯對人類牙髓細胞的毒性可能是來自活性含氧物生成的增加，造成DNA的損傷而改變細胞週期調控因子的表現，使細胞週期受到干擾，最終抑制細胞的生長和造成細胞的死亡。另外，10-(2-甲基丙烯酰氧基)磷酸單癸酯所造成的氧化壓力上升，可能也會使發炎相關因子表現增加而啟動發炎反應。這些結果有助於我們了解10-(2-甲基丙烯酰氧基)磷酸單癸酯的毒性作用以及未來牙本質黏著劑的改良。 | zh_TW |
| dc.description.abstract | Aim: 10-MDP (10-Methacryloyloxydecyl Dihydrogen Phosphate), a functional resin monomer, has been widely added in universal adhesives but the cytotoxicity and genotoxicity are poorly understood. This study aimed to investigate the toxic effect of 10-MDP and the mechanism of it through analyzing the cell viability, production of reactive oxygen species, inflammatory response and cell cycle distribution. Then the mechanism of the 10-MDP toxicity was surveyed by analyzing expression of the extracellular matrix protein, oxidative stress related enzymes, cell cycle related molecules and inflammatory mediators. Material and methods: Primary human dental pulp cells were treated with different concentrations of 10-MDP (0, 10, 50, 100, 250, 500 μM). The cell viability was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. The cell cycle distribution and the production of reactive oxygen species were analyzed by flow cytometry with propidium iodide and 2', 7'-dichlorofluorescin diacetate staining respectively. Real-time quantitative PCR, western blot and immunofluorescence staining were used to evaluate the expression of extracellular matrix protein (collagen I), oxidative stress related enzymes(Nrf2, p-Nrf2 and HO-1), inflammatory mediators(COX-2, IL-6 and IL-8), and cell cycle related molecules (cyclin B1, cdc25C, p21, GADD45α and p-ATM). Results: 10-MDP induced morphological changes of human dental pulp cells and decreased cell viability by 57 – 81% at high concentrations of 250 and 500 μM. The production of reactive oxygen species was increased after exposure to 250 μM 10-MDP for 24 hours, and the expression of p-Nrf2, HO-1, COX-2, IL-6 and IL-8 was elevated. Moreover, the cell cycle was disturbed, showing increased sub-G0/G1, G0/G1 and G2/M cell percentages. The expression of p-ATM, p21 and GADD45α was up-regulated and the expression of collagen I, cyclin B1 and cdc25C was inhibited after exposure to 10-MDP 250 μM for 24 hours. Conclusions: The results of this study give an insight of the toxic effect and its mechanism of 10-MDP toward human dental pulp cells. The 10-MDP-induced toxicity may cause an elevated oxidative stress due to overproduction of reactive oxygen species. The increased oxidative stress might change the expression of cell cycle regulatory molecules, resulting cell cycle disturbance. Moreover, 10-MDP might stimulate expression of inflammatory mediators, causing inflammatory response. These findings may be helpful in understanding the toxic effect of 10-MDP and the improvement of dental adhesives in the future. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T23:00:46Z (GMT). No. of bitstreams: 1 U0001-1107202220233300.pdf: 2894807 bytes, checksum: d7a9a0f328379e25caa95107b9d5a663 (MD5) Previous issue date: 2022 | en |
| 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 第一章 文獻回顧 1 1.1 序言 1 1.1.1 牙科黏著劑(Dental adhesives) 1 1.1.2 功能性單體(Functional monomer) 2 1.1.3 10-(2-甲基丙烯酰氧基)磷酸單癸酯 (10-MDP) 3 1.2 對細胞毒性(Cytotoxicity)以及對細胞週期(Cell cycle)之影響 4 1.2.1 10-MDP之細胞毒性 4 1.2.2 細胞外基質(Extracellular matrix) 4 1.2.3 細胞週期(Cell cycle) 5 1.2.4 細胞週期之調控蛋白(Cell cycle regulatory protein) 6 1.2.5 細胞週期檢查點(Cell cycle checkpoints) 7 1.3 對氧化壓力之影響 (Oxidative stress) 9 1.3.1 活性含氧物 (Reactive oxygen, species, ROS) 9 1.3.2 Nuclear factor erythroid 2-related factor 2 (Nrf2) 10 1.3.3 血鐵質氧化酶-1 (Heme oxygenase-1， HO-1) 11 1.4 對發炎反應之影響 (Inflammatory process) 11 1.4.1 環氧化酶-2 (Cyclooxygenase-2，COX-2) 12 1.4.2 白細胞介素-6 (Interleukin-6，IL-6) 13 1.4.3 白細胞介素-8 (Interleukin-8，IL-8) 13 第二章 實驗目的與假說 15 第三章 材料與方法 16 3.1 化學藥物的準備 16 3.2 人類牙髓細胞(human dental pulp cells，hDPCs)的培養 17 3.3 細胞存活率分析 (MTT assay) 18 3.4 流式細胞術 (Flow cytometry) 18 3.4.1 細胞週期分析(Cell cycle analysis) 18 3.4.2 活性含氧物(Reactive oxygen species, ROS)的生成之分析 19 3.5 即時定量聚合酶連鎖反應 (Real-time quantitative PCR) 20 3.5.1 核糖核酸的萃取(RNA extraction) 20 3.5.2 RNA定量(RNA quantification) 22 3.5.3 反轉錄(Reverse transcription) 22 3.5.4 及時定量聚合酶連鎖反應 23 3.6 西方墨點法 (Western blot) 23 3.6.1 蛋白質萃取(Protein extraction) 23 3.6.2 蛋白質定量 (Protein quantification) 24 3.6.3 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 25 3.6.4 轉漬(Transfer gel) 25 3.6.5 阻斷以及抗體反應(Blocking and antibody hybridization) 26 3.6.6 化學冷光影像擷取(Chemiluminescence photography) 26 3.7 免疫螢光染色 (Immunofluorescence assay) 26 3.7.1 細胞培養 26 3.7.2 免疫螢光染色 (Immunofluorscence staining) 27 3.8 統計分析 28 第四章 實驗結果 29 4.1 10-MDP濃度對於hDPCs細胞存活率的影響：MTT assay 29 4.2 10-MDP濃度對於hDPCs型態的影響 29 4.3 10-MDP對於Collagen I基因和蛋白表現之影響 29 4.4 10-MDP濃度對於hDPCs細胞週期的影響 30 4.4.1 對於細胞週期運行的影響：Flow cytometry PI staining 30 4.4.2對於細胞週期調控基因和蛋白的影響 30 4.5 10-MDP濃度對於hDPCs氧化壓力的影響 31 4.5.1 對於活性含氧物(ROS)生成的影響：Flow cytometry DCF staining 31 4.5.2 對於Nrf2的基因和蛋白表現的影響 31 4.5.3 對於HO-1的基因和蛋白表現的影響 32 4.6 10-MDP濃度對於hDPCs發炎反應的影響 32 4.6.1 對於COX-2的基因和蛋白表現的影響 32 4.6.2 對於IL-6的基因和蛋白表現的影響 32 4.6.3 對於IL-8的基因和蛋白表現的影響 33 第五章 討論 34 第六章 結論 40 參考文獻 41 | |
| dc.language.iso | zh-TW | |
| dc.subject | 人類牙髓細胞 | zh_TW |
| dc.subject | 發炎反應 | zh_TW |
| dc.subject | 氧化壓力 | zh_TW |
| dc.subject | 細胞週期 | zh_TW |
| dc.subject | 10-(2-甲基丙烯酰氧基)磷酸單癸酯 | zh_TW |
| dc.subject | oxidative stress | en |
| dc.subject | 10-MDP | en |
| dc.subject | inflammatory response | en |
| dc.subject | human dental pulp cells | en |
| dc.subject | cell cycle | en |
| dc.title | 10-(2-甲基丙烯酰氧基)磷酸單癸酯對於人類牙髓細胞之毒性效應及相關機制 | zh_TW |
| dc.title | Toxic Effect of 10-Methacryloyloxydecyl Dihydrogen Phosphate(10-MDP) on Human Dental Pulp Cells and Related Mechanisms | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張美姬(Mei-Chi Chang) | |
| dc.subject.keyword | 10-(2-甲基丙烯酰氧基)磷酸單癸酯,人類牙髓細胞,細胞週期,氧化壓力,發炎反應, | zh_TW |
| dc.subject.keyword | 10-MDP,human dental pulp cells,cell cycle,oxidative stress,inflammatory response, | en |
| dc.relation.page | 77 | |
| dc.identifier.doi | 10.6342/NTU202201410 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-07-21 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 臨床牙醫學研究所 | zh_TW |
| dc.date.embargo-lift | 2022-10-03 | - |
| 顯示於系所單位： | 臨床牙醫學研究所 | |
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