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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張曉華 | zh_TW |
dc.contributor.advisor | Hsiao-Hua Chang | en |
dc.contributor.author | 石婉妤 | zh_TW |
dc.contributor.author | Wan-Yu Shih | en |
dc.date.accessioned | 2023-09-22T16:08:36Z | - |
dc.date.available | 2023-11-09 | - |
dc.date.copyright | 2023-09-22 | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2023-07-20 | - |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89794 | - |
dc.description.abstract | 目的:功能性單體4-甲基丙烯酰氧基偏苯三酸酐(4-methacryloxyethyl trimellitic anhydride, 4-META)被廣泛使用於牙科樹脂黏著劑(resin adhesives)中,這些樹脂材料填充在牙齒中、接觸牙齒表面,可能面臨聚合不完全、長期下來於口腔環境中水解的可能,過去的研究已證實許多牙科材料常見的樹脂單體都具有細胞毒性,會導致DNA破壞、細胞氧化壓力上升、干擾細胞週期運行、造成細胞死亡、誘導發炎反應等。然而,迄今為止對於4-META的研究相當匱乏,憑現有的研究我們仍無法了解4-META對於人類牙髓細胞的毒性及可能造成的影響。本研究目的在探討4-META對於人類牙髓細胞所造成的影響,包括:細胞活性、細胞週期的運行、細胞凋亡、氧化壓力及發炎反應的變化,並進一步研究調控細胞週期、氧化壓力、細胞凋亡及發炎反應相關之基因與蛋白質的表現,希望能了解4-META造成毒性的機制。
實驗方法:本實驗使用體外培養的初代人類牙髓細胞,加入0、0.5、1、 2.5、5及7.5 mM的4-META培養24小時,以MTT檢測細胞存活率,使用流式細胞術以PI分析細胞週期、以PI與Annexin V雙染分析細胞凋亡、以DCF螢光染色分析細胞活性氧的生成、以CMF螢光染色分析細胞穀胱甘肽的生成,並以即時定量聚合酶連鎖反應、西方點墨法及免疫化學螢光染色來檢測細胞週期調控因子(cyclin B1、cdc25C、cdc2、p21、p53、GADD45α)、細胞凋亡調控因子(BAX)、氧化壓力調控因子(Nrf2、HO-1)、發炎調控因子(COX-2、IL-6、IL-8)的基因與蛋白質表現之變化。 實驗結果:當人類牙髓細胞暴露在2.5 mM的4-META時會導致細胞存活率降低、細胞形態產生改變,本實驗測得的半抑制濃度應介於2.5 mM - 5 mM之間。在2.5 mM的4-META會使人類牙髓細胞的活性氧生成增加、並產生S phase停滯。當4-META濃度上升至5及7.5 mM時,GSH濃度下降、細胞凋亡的比例、sub G0/G1比例顯著增加。高濃度的4-META會使細胞週期調控因子的表現cdc2、cyclin B1、cdc25C表現下降,p21、p53、GADD45α表現上升。4-META會提高氧化壓力調控因子Nrf2、HO-1、細胞凋亡調控因子BAX、以及發炎相關因子COX-2、IL-6、IL-8的表現。 結論:由本實驗的結果,我們推測4-META造成人類牙髓細胞的毒性其成因是由於活性氧生成量增加,導致細胞的氧化壓力上升,進而造成DNA的受損而改變細胞週期調控因子的表現,擾亂細胞週期的運行。而隨著4-META濃度上升,細胞無法於細胞週期停滯時完成修復、穀胱甘肽的耗盡、細胞凋亡與壞死增加。而當氧化壓力上升,會活化Nrf2,因而提高HO-1的表現量。4-META造成的毒性也會使發炎相關因子的表現上升,誘導發炎反應。就目前我們的研究結果可以初步瞭解人類牙髓組織在接觸到4-META後會造成的影響。但對於4-META造成氧化壓力上升、干擾細胞週期、及如何誘導發炎等機制尚有許多未了解之處,還待未來更進一步的研究,希望未來能更了解這些機制,更能有助於臨床上的應用與牙科材料的改良。 | zh_TW |
dc.description.abstract | Aim: As a functional monomer, 4-methacryloxyethyl trimellitic anhydride (4-META) is widely used in dental resin adhesives. These resin materials which are in long-term contact with dental tissues may face potential issues such as incomplete polymerization and hydrolysis, thus cause monomer releasing into the oral environment consequently. Previous studies have confirmed that many common resin monomers in dental materials have cytotoxicity, leading to DNA damage, increased cellular oxidative stress, disturbance of cell cycle progression, cell death, and induced inflammatory reactions. However, research on 4-META has been scarce so far. Based on existing few studies, the cytotoxicity and potential effects of 4-META on human dental pulp cells are poorly understood. The aim of this study is to investigate the effects of 4-META on human dental pulp cells, including cell viability, cell cycle progression, cell apoptosis, production of reactive oxygen species, and inflammatory responses. Furthermore, in order to understand the mechanisms of the toxicity caused by 4-META, the expression of some important factors regulating cell cycle, oxidative stress, apoptosis, and inflammation were also analyzed.
Material and methods: In this experiment, primary human dental pulp cells were cultured in vitro and treated with final concentrations of 0, 0.5, 1, 2.5, 5, and 7.5 mM of 4-META for 24 hours. Cell viability was assessed using the MTT assay. Flow cytometry with propidium iodide (PI) staining was used to analyze cell cycle distribution, while dual staining with PI and Annexin V was used to analyze cell apoptosis. The production of reactive oxygen species was analyzed using DCF fluorescence staining, and the production of cellular glutathione was assessed using CMF fluorescence staining. Real-time quantitative polymerase chain reaction (PCR), Western blotting, and immunofluorescence staining were performed to examine the changes in gene and protein expression of cell cycle regulatory factors (cyclin B1, cdc25C, cdc2, p21, p53, GADD45α), apoptosis related factor (BAX), oxidative stress regulatory factors (Nrf2, HO-1), and inflammatory related factors (COX-2, IL-6, IL-8). Results: Exposure of human dental pulp cells to 2.5 mM of 4-META resulted in decreased cell viability and cell morphological changes. At 2.5 mM of 4-META, the production of reactive oxygen species in human dental pulp cells elevated, and S phase cell cycle arrest occurred. When the concentration of 4-META increased to 5 mM and 7.5 mM, the glutathione concentration decreased, the proportion of cell apoptosis and sub G0/G1 phase raised significantly. High concentrations of 4-META lead to a decrease in the expressions of cdc2, cyclin B1, and cdc25C, while the expressions of p21, p53, and GADD45α were increased. 4-META also enhanced Nrf2, HO-1, and the apoptosis related factor BAX. It also enhances the expression of pro-inflammatory factors such as COX-2, IL-6, and IL-8. Conclusions: The present in vitro experiment demonstrated that the toxicity of 4-META on human dental pulp cells may be related to the increase of reactive oxygen species, leading to elevated oxidative stress and subsequent DNA damage, which alters the expression of cell cycle regulatory factors and disturbs cell cycle progression. As the concentration of 4-META increases, cells may not be able to repair DNA completely during cell cycle arrest, leading to increased cell apoptosis and necrosis. 4-META may also trigger inflammatory responses by inducing the expression of inflammatory related factors. The results of this study provide a preliminary understanding of the toxic effect and mechanism of 4-META on human dental pulp tissue. Further studies are needed to verify the mechanisms of the toxicity of 4-META and develop safer materials in clinical use. | en |
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dc.description.provenance | Made available in DSpace on 2023-09-22T16:08:36Z (GMT). No. of bitstreams: 0 | 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 viii 附錄 xiv 表格 xv 附圖 xvi 第一章 文獻回顧 1 1.1 序言 1 1.1.1 牙科樹脂(Dental resin) 1 1.1.2 樹脂單體(Resin monomer) 2 1.2 4-甲基丙烯酰氧基偏苯三酸酐 (4-methacryloxyethyl trimellitic anhydride, 4-META) 2 1.2.1 4-甲基丙烯酰氧基偏苯三酸酐介紹 2 1.2.2 4-META之細胞毒性 4 1.3 對細胞週期(Cell cycle)之影響 5 1.3.1 細胞週期(Cell cycle) 5 1.3.2 細胞週期之調控蛋白(Cell cycle regulatory protein) 6 1.3.3 細胞週期檢查點(Cell cycle checkpoints) 7 1.4 細胞死亡途徑(Cell death pathway):細胞凋亡(Apoptosis) 8 1.5 對氧化壓力之影響(Oxidative stress) 10 1.5.1 活性含氧物(Reactive oxygen, species, ROS) 10 1.5.2 穀胱甘肽(Glutathione, GSH) 11 1.5.3 Nuclear factor erythroid 2-related factor 2 (Nrf2) 12 1.5.4 血鐵質氧化酶-1(Heme oxygenase-1, HO-1) 12 1.6 對發炎反應之影響(Inflammation) 13 1.6.1 牙髓的發炎反應(Inflammatory response of the dental pulp) 13 1.6.2 環氧化酶-2(Cyclooxygenase-2,COX-2) 14 1.6.3 白細胞介素-6(Interleukin-6,IL-6) 15 1.6.4 白細胞介素-8(Interleukin-8,IL-8) 15 第二章 實驗目的與假說 17 第三章 材料與方法 18 3.1 材料準備 18 3.1.1 樣本試劑 18 3.1.2 儀器設備 19 3.2 人類牙髓細胞培養 20 3.3 細胞存活率分析:MTT Assay 21 3.4 流式細胞術(Flow Cytometry) 21 3.4.1 細胞週期分析(Cell cycle analysis) 21 3.4.2 細胞凋亡檢測(Apoptosis detection):Annexin V-FITC / PI assay 22 3.4.3 活性含氧物(Reactive oxygen species, ROS)生成分析(Production of ROS) 24 3.4.4 穀胱甘肽生成分析(Production of glutathione) 24 3.5 即時定量聚合酶連鎖反應(Real-time Quantitative PCR) 25 3.5.1 核糖核酸萃取(RNA extraction) 25 3.5.2 核糖核酸的定量(RNA quantification) 27 3.5.3 反轉錄(Reverse transcription) 27 3.5.4 即時定量聚合酶連鎖反應(Real-time Quantitative PCR) 28 3.6 西方墨點法(Western blot) 28 3.6.1 蛋白質萃取(Protein extraction) 29 3.6.2 蛋白質定量(Protein quantification) 29 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) 30 3.6.4 轉漬(Transfer gel) 30 3.6.5 阻斷及抗體雜合(Blocking and antibody hybridization) 31 3.6.6 化學發光影像擷取(Chemiluminescence photography) 31 3.7 免疫化學螢光染色(Immunofluorescence Assay) 32 3.8 統計分析 33 第四章 實驗結果 34 4.1 4-META濃度對於人類牙髓細胞存活率的影響:MTT assay 34 4.2 4-META濃度對於人類牙髓細胞型態的影響 34 4.3 4-META濃度對於人類牙髓細胞之細胞週期運行的影響 34 4.3.1 對於細胞週期進程的影響:Flow cytometry PI staining 34 4.3.2 對於cdc25C基因及蛋白質表現的影響 35 4.3.3 對於cdc2基因及蛋白質表現的影響 35 4.3.4 對於Cyclin B1基因及蛋白質表現的影響 36 4.3.5 對於GADD45α基因及蛋白質表現的影響 36 4.3.6 對於p21基因及蛋白質表現的影響 36 4.3.7 對於p53基因及蛋白質表現的影響 37 4.4 4-META濃度對於人類牙髓細胞之細胞死亡途徑(cell death pathway)的影響 37 4.4.1 對於細胞死亡途徑的影響:Annexin V-FITC / PI assay 37 4.4.2 對於BAX基因及蛋白質表現的影響 38 4.5 4-META濃度對於人類牙髓細胞之氧化壓力的影響 38 4.5.1 對於ROS生成的影響:DCF fluorescence flow cytometry 38 4.5.2 對於細胞中穀胱甘肽(glutathione, GSH)生成的影響:CMF fluorescence flow cytometry 38 4.5.3 對於Nrf2基因及蛋白質表現的影響 39 4.5.4 對於HO-1基因及蛋白質表現的影響 39 4.6 4-META濃度對於人類牙髓細胞的發炎反應之影響 39 4.6.1 對於COX-2基因及蛋白質表現的影響 39 4.6.2 對於IL-8基因及蛋白質表現的影響 40 4.6.3 對於IL-6基因及蛋白質表現的影響 40 第五章 討論 41 5.1 對於人類牙髓細胞存活率及細胞型態之影響 41 5.2 對於人類牙髓細胞的細胞週期進程之影響 43 5.3 對於人類牙髓細胞的細胞死亡途徑之影響 45 5.4 對於人類牙髓細胞的氧化壓力之影響 46 5.5 對於人類牙髓細胞的發炎反應之影響 47 第六章 結論 50 參考文獻 51 附錄1 4-META的化學結構 60 附錄2 細胞週期以及不同階段的Cyclin/CDK complex示意圖 61 附錄3 哺乳動物細胞週期的檢查點機制圖 62 表格1 即時定量聚合酶連鎖反應所使用的引子(primer)之序列(sequence) 63 表格2 西方墨點法所使用的蛋白質萃取緩衝溶液(protein extraction buffer)製備方法 64 表格3 SDS-PAGE膠體的製備 65 表格4 西方點墨法緩衝液的製備 66 表格5 西方墨點法以及免疫螢光染色所使用的一級抗體 67 表格6 在不同濃度的4-META下培養24小時後對於人類牙髓細胞存活率的影響 68 表格7 在不同濃度的4-META下培養24小時後對於人類牙髓細胞週期的影響 69 表格8 在不同濃度的4-META下培養24小時後對於人類牙髓細胞死亡途徑的影響 70 圖1 4-META對於人類牙髓細胞的存活率之影響:MTT assay 71 圖2 4-META對於人類牙髓細胞的細胞型態之影響 72 圖3 4-META對於人類牙髓細胞的細胞週期之影響:PI staining flow cytometry 73 圖4 4-META對於人類牙髓細胞的cdc25C表現之影響 75 圖5 4-META對於人類牙髓細胞的cdc2表現之影響 76 圖6 4-META對於人類牙髓細胞的Cyclin B1表現之影響 77 圖7a 4-META對於人類牙髓細胞的GADD45⍺ mRNA表現之影響 78 圖7b-g 4-META對於人類牙髓細胞的GADD45⍺蛋白質表現之影響 79 圖8 4 -META對於人類牙髓細胞的p21表現之影響 80 圖9 4-META對於人類牙髓細胞的p53表現之影響 81 圖10 4-META對於人類牙髓細胞的細胞死亡途徑之影響:Annexin V-FITC / PI assay 82 圖11a 4-META對於人類牙髓細胞的BAX mRNA表現之影響 84 圖11b-g 4-META對於人類牙髓細胞的BAX蛋白質表現之影響 85 圖12 4-META對於人類牙髓細胞的ROS 生成量之變化:DCF fluorescence 86 圖13 4-META對於人類牙髓細胞的穀胱甘肽(glutathione)生成量之變化:CMF fluorescence 88 圖14a 4-META對於人類牙髓細胞Nrf2的mRNA表現之影響 90 圖14b-g 4-META對於人類牙髓細胞Nrf2的蛋白質表現之影響 91 圖15 4-META對於人類牙髓細胞HO-1的mRNA及蛋白質表現之影響 92 圖16a 4-META對於人類牙髓細胞的COX-2 mRNA表現之影響 93 圖16b-g 4-META對於人類牙髓細胞的COX-2 蛋白質表現之影響 94 圖17 4-META對於人類牙髓細胞的IL-8表現之影響 95 圖18 4-META對於人類牙髓細胞的IL-6表現之影響 96 圖19 4-META對於人類牙髓細胞的影響之機制圖 97 | - |
dc.language.iso | zh_TW | - |
dc.title | 4-甲基丙烯酰氧基偏苯三酸酐對人類牙髓細胞的毒性反應研究 | zh_TW |
dc.title | Investigation of the Toxic Effect of 4-Methacryloxyethyl Trimellitic Anhydride (4-META) on Human Dental Pulp Cells | en |
dc.type | Thesis | - |
dc.date.schoolyear | 111-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.coadvisor | 鄭景暉 | zh_TW |
dc.contributor.coadvisor | Jiiang-Huei Jeng | en |
dc.contributor.oralexamcommittee | 張美姬;王姻麟 | zh_TW |
dc.contributor.oralexamcommittee | Mei-Chi Chang;Yin-Lin Wang | en |
dc.subject.keyword | 4-甲基丙烯酰氧基偏苯三酸酐,人類牙髓細胞,細胞毒性,細胞週期,細胞凋亡,氧化壓力,發炎反應, | zh_TW |
dc.subject.keyword | 4-methacryloxyethyl trimellitic anhydride,human dental pulp cell,cell cycle progression,cell death pathway,apoptosis,inflammatory reactions,oxidative stress, | en |
dc.relation.page | 97 | - |
dc.identifier.doi | 10.6342/NTU202301311 | - |
dc.rights.note | 同意授權(限校園內公開) | - |
dc.date.accepted | 2023-07-20 | - |
dc.contributor.author-college | 醫學院 | - |
dc.contributor.author-dept | 臨床牙醫學研究所 | - |
顯示於系所單位: | 臨床牙醫學研究所 |
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