請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32963
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭景暉 | |
dc.contributor.author | Min-Tsz Wu | en |
dc.contributor.author | 吳敏慈 | zh_TW |
dc.date.accessioned | 2021-06-13T04:20:20Z | - |
dc.date.available | 2012-10-05 | |
dc.date.copyright | 2011-10-05 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-07-27 | |
dc.identifier.citation | 1. Craig RG and Powers JM, Restorative dental materials 11th edition, Ch7 & 9.
2. Masuki K, Nomura Y, Bhawal UK, Sawajiri M, Hirata I, Nahara Y and Okazaki M, Apoptotic and necrotic influence of dental resin polymerization initiators in human gingival fibroblast cultures. Dental Materials Journal 2007; 26: 861-869. 3. Spahl W, Budzikiewicz H and Geurtsen W, Determination of leachable components from four commercial dental composites by gas and liquid chromatography/mass spectrometry. Journal of Dentistry 1998; 26: 137-145. 4. Michelsen VB, Moe G, Skålevik R, Jensen E and Lygre H, Quantification of organic elutes from polymerized resin-based dental restorative materials by use of GC/MS. Journal of Chromatography B 2007; 850: 83-91. 5. Stansbury JW, Curing dental resins and composites by photopolymerization. Journal of Esthetic Dentistry 2000; 12: 300-308. 6. Taira M, Urabe H, Hirose T, Wakasa K and Yamaki M, Analysis of photo-initiators in visible-light-cured dental composite resins. Journal of Dental Research 1988; 67: 24-28. 7. Volk J, Ziemann C, Leyhausen G and Geurtsen W, Non-irradiated camphorquinone induces DNA damage in human gingival fibroblasts. Dental Materials 2009; 25: 1556-1563. 8. Geurtsen W, Lehmann F, Spahl W and Leyhausen G, Cytotoxicity of 35 dental resin composite monomers/additives in permanent 3T3 and three human primary fibroblast cultures. Journal of Biomedical Materials Research 1998; 41: 474-480. 9. Atsumi T, Murata J, Kamiyanagi I, Fujisawa S and Ueha T, Cytotoxicity of photosensitizers camphorquinone and 9-fluorenone with visible light irradiation on a human submandibular-duct cell line in vitro. Archives of Oral Biology 1998; 43:73-81. 10. Atsumi T, Iwakura I, Fujisawa S and Ueha T, The production of reactive oxygen species by irradiated camphorquinone-related photosensitizer and their effect on cytotoxicity. Archives of Oral Biology 2001; 46: 391-401. 11. Atsumi T, Ishihara M, Kadoma Y, Tonosaki K and Fujisawa S, Comparative radical production and cytotoxicity induced by camphorquinone and 9-fluorenone against human pulp fibroblasts. Journal of Oral Rehabilitation 2004; 31: 1155-1164. 12. Hanks CT, Strawn SE, Wataha JC and Craig RG, Cytotoxic effects of resin components on cultured mammalian fibroblasts. Journal of Dental Research 1991; 70: 1450-1455. 13. Engelmann J, Volk J, Leyhausen G and Geurtsen W, ROS formation and glutathione levels in human oral fibroblasts exposed to TEGDMA and camphorquinone. Journal of Biomedical Materials Research Part B: Applied Biomaterials 2005; 75B: 272-276. 14. Pagoria D and Geurtsen W, The effect of N-acetyl-L-cysteine and ascorbic acid on visible-light-irradiated camphorquinone/ N,N-dimethyl-ρ-toluidine- induced oxidative stress in two immortalized cell lines. Biomaterials 2005; 26: 6136-6142. 15. Li YC, Huang FM, Lee SS, Lin RH and Chang YC, Protective effects of antioxidants on micronuclei induced by camphorquinone/ N,N-dimethyl-ρ-toluidine employing in vitro mammalian test system. Journal of Biomedical Materials Research Part B: Applied Biomaterials 2007; 82B: 23-28. 16. Datar RA, Rueggeberg FA, Caughman GB, Wataha JC, Lewis JB and Schuster GS, Effects of sub-toxic concentrations of camphorquinone on cell lipid metabolism. Journal of Biomaterials Science, Polymer Edition 2005; 16: 1293-1302. 17. Vermeulen K, Bockstaele DRV and Berneman ZN, The cell cycle: a review ofregulation, deregulation and therapeutic targets in cancer. Cell Proliferation 2003; 36: 131-149. 18. Shackelford RE, Kaufmann WK and Paules RS, Oxidative stress and cell cycle checkpoint function. Free Radical Biology & Medicine 2000; 28: 1387-1404. 19. Murray A and Hunt T, The cell cycle: an introduction. New York: W. H. Freeman and Co. 1993. 20. Jung YS, Qian Y and Chen X, Examination of the expanding pathways for the regulation of p21 expression and activity. Cellular Signalling 2010; 22: 1003-1012. 21. Taylor WR and Stark GR, Regulation of the G2/M transition by p53. Oncogene 2001; 20: 1803-1815. 22. Nyberg KA, Michelson RJ, Putnam CW and Weinert TA, Toward maintaining the genome: DNA damage and replication checkpoints. Annual Review of Genetics 2002; 36: 617-656. 23. Shiloh Y, ATM and related protein kinases: safeguarding genome integrity. Nature Reviews Cancer 2003; 3: 155-168. 24. Zhou BBS and Elledge SJ, The DNA damage response: putting checkpoints in perspective. Nature 2000; 408: 433-439. 25. Bartek J and Lukas J, Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 2003; 3: 421-429. 26. Lukas J, Lukas C and Bartek J, Mammalian cell cycle checkpoints: signaling pathways and their organization in space and time. DNA Repair 2004; 3: 997-1007. 27. Majno G and Joris I, Apoptosis, oncosis, and necrosis. An overview of cell death. American Journal of Pathology 1995; 146: 3-15. 28. Wyllie AH, “Where, o death, is thy sting?” A brief review of apoptosis biology. Molecular Neurobiology 2010; 42: 4-9. 29. Edited by Wilson JW, Booth C and Pottern CS, Apoptosis genes. 1998. 30. Kikuchi G, Yoshida T and Noguchi M, Heme oxygenase and heme degradation. Biochemical and Biophysical Research Communications 2005; 338: 558-567. 31. Chung SW, Hall SR and Perrella MA, Role of haem oxygenase-1 in microbial host defence. Cellular Microbiology 2009; 11: 199-207. 32. Chan CP, Lan WH, Chang MC, Chen YJ, Lan WC, Chang HH and Jeng JH, Effects of TGF-βs on the growth, collagen synthesis and collagen lattice contraction of human dental pulp fibroblasts in vitro. Archives of Oral Biology 2005; 50: 469-479. 33. Tai TF, Chan CP, Lin CC, Chen LI, Jeng JH and Chang MC, Transforming growth factor beta 2 regulates growth and differentiation of pulp cells via alk5/smad 2/3. Journal of Endodontics 2008; 34: 427-432. 34. Mosmann T, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 1983; 65: 55-63. 35. Agnihotri N and Mishra PC, Mechanism of scavenging action of N-acetylcysteine for the OH radical: a quantum computational study. Journal of Physical Chemistry B 2009; 113: 12096-12104. 36. Chelikani P, Fita I and Loewen PC, Diversity of structures and properties among catalases. Cellular and Molecular Life Sciences 2004; 61: 192-208. 37. Labbe RF, Vreman HJ and Stevenson DK, Zinc protoporphyrin: a metabolite with a mission. Clinical Chemistry 1999; 45: 2060-2072. 38. Current protocols in cytometry, 2008. Ch4 Molecular and cellular probes. 39. Krishan A, Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. The Journal of Cell Biology 1975; 66: 188-193. 40. McClay DR. The role of thin filopodia in motility and morphogenesis. ExperimentalCell Research 1999; 253: 296-301. 41. Sheetz MP, Wayne DB and Pearlman AL, Extension of filopodia by motor-dependent actin assembly. Cell Motility and the Cytoskeleton 1992; 22: 160-169. 42. Janke V, Neuhoff NV, Schlegelberger B, Leyhausen G and Geurtsen W, TEGDMA causes apoptosis in primary human gingival fibroblasts. Journal of Dental Research 2003; 82: 814-818. 43. Chang HH, Guo MK, Kasten FH, Chang MC, Huang GF, Wang YL, Wang RS and Jeng JH, Stimulation of glutathione depletion, ROS production and cell cycle arrest of dental pulp cells and gingival epithelial cells by HEMA. Biomaterials 2005; 26: 745-753. 44. Cakir Y and Ballinger SW, Reactive species-mediated regulation of cell signaling and the cell cycle: the role of MAPK. Antioxidants & Redox Signaling 2005; 7: 726-735. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32963 | - |
dc.description.abstract | 實驗目的:樟腦醌(Camphorquinone, CQ)是目前複合樹脂中最常被使用的感光劑。本實驗的目的在於探討CQ對人類牙髓細胞的細胞毒性,以及對細胞週期和細胞凋亡相關基因與蛋白表現的影響,並確認其毒性與活性氧(ROS)形成的關聯性。
實驗方法:將人類牙髓細胞暴露於不同濃度的CQ (0.1-2 mM)之下培養後,以顯微鏡觀察細胞形態變化,以MTT測定來評估細胞存活,並以流式細胞分析技術(flow cytometry)進行細胞週期、細胞死亡及活性氧形成等分析。基因與蛋白表現的變化則分別以反轉錄聚合酶連鎖反應(RT-PCR)和西方墨點法(western blot)來觀察。至於分析活性氧與HO-1所扮演的角色,是將經可清除活性氧的NAC (1 mM)及catalase (2000 U/ml)或HO抑制劑ZnPP (2.5及5 μM)前處理30分鐘的牙髓細胞暴露於CQ (2 mM)之下培養後,以MTT測定來觀察細胞存活的變化。最後以單因子變異數分析搭配Tukey事後檢定來分析數據。 實驗結果:人類牙髓細胞經濃度1 mM和2 mM的CQ作用24小時後,可觀察到細胞形態改變和細胞存活率降低的情形(分別是70%與50%)。在這樣的濃度下,CQ造成G2/M期的細胞週期停滯,同時相關基因和蛋白中cdc2、cyclin B、p-cdc2及cdc25C的表現被抑制,p21及p-p53 則被促進。細胞死亡方面,1 mM的CQ處理會造成凋亡的細胞數目增加,而當CQ濃度增加到2 mM時,凋亡、壞死及先凋亡後壞死的細胞數目皆有顯著增加。在細胞凋亡的相關基因和蛋白中,Bax的表現被促進,Bcl2則被抑制。此外,人類牙髓細胞暴露於濃度超過0.5 mM的CQ中3小時後,便可觀察到顯著的活性氧增加,24小時後也能看到HO-1表現的上升,而經NAC或catalase前處理的細胞中CQ所造成的細胞存活率降低會被抑制,至於ZnPP (5 μM)則會促進CQ所造成的細胞存活率降低。 結論:CQ濃度高於0.5 mM時便會造成活性氧的生成顯著增加,而明顯的細胞毒性,則發生於濃度超過1 mM後。CQ在超過1 mM的濃度下會造成細胞形態改變、存活率降低、G2/M期的細胞週期停滯以及細胞死亡,特別是凋亡。這些變化可能與活性氧產生有關,進而造成許多基因(如:cdc2、cyclin B、cdc25C、p21、Bax以及Bcl2)表現的改變,至於HO-1表現的增加可能扮演了保護細胞的角色,值得進一步研究加以證實。雖然CQ在樹脂中的含量不高,但當殘餘CQ擴散到體積相對較小的牙髓腔時,滲出物的濃度片有機會高到足以對牙髓細胞造成傷害的程度,因此在臨床上以樹脂進行齲齒填補時,應適時使用基底材料來保護牙髓。 | zh_TW |
dc.description.abstract | Aim: Camphorquinone (CQ) is the primarily used photosensitizer in resin composites nowadays. The purpose of this study is to investigate the influences of CQ on cytotoxicity to human dental pulp cells. Then, its effects on the expression of cell cycle and apoptosis related genes and proteins are evaluated. Besides, the relationship between ROS formation and its toxicity is also observed.
Materials and methods: Primary-cultured human dental pulp cells were treated with different concentrations of CQ (0.1 to 2 mM). Cell morphology was observed under a phase contrast microscope. Cell proliferation was evaluated by MTT assay. Cell cycle analysis, cell death pattern and ROS formation were investigated by flow cytometry. Changes in mRNA expression were determined by reverse-transcription polymerase chain reaction (RT-PCR). Changes in protein production were evaluated by western blot. As for evaluation of the roles of ROS and HO-1, pulp cells were pre-treated for 30 minutes with NAC (1 mM) and catalase (2000 U/ml) which can remove ROS, or HO inhibitor ZnPP (2.5 and 5 μM) before co-incubation with 2 mM CQ. Then, MTT assay was used to investigate the changes of cell viability. One-way ANOVA and post hoc Tukey test was used to analyze differences between experimental and control groups. Results: In human dental pulp cells, CQ induced morphological changes and a significant decrease of cell viability, to about 70% and 50% respectively, at the concentrations of 1 mM and 2 mM after incubation for 24 hours. At these concentrations CQ led to G2/M cell cycle arrest. The expression or production of cdc2, cyclin B, p-cdc2 and cdc25C was inhibited, while that of p21 and p-p53 was promoted. 1 mM CQ caused an increase of apoptotic cells, and at the concentration of 2 mM, obvious increases of apoptotic, necrotic as well as apoptotic/necrotic cells were observed. In the same time, the expression and production of Bax was promoted, while that of Bcl2 was inhibited. Besides, exposure to CQ higher than 0.5 mM for 3 hours caused a dose-dependent increase of ROS, and an increase of HO-1 expression was noted after 24 hours. The reduction of cell viability caused by CQ can be inhibited by NAC or catalase pre-treatment, and can be promoted by 5 μM ZnPP pre-treatment. Conclusions: CQ at a concentration higher than 0.5 mM caused a marked production of ROS, and a significant cytotoxicity was noted at a concentration above 1 mM. Under the concentrations higher than 1 mM, CQ can cause changes of cell morphology, reduction of cell viability, G2/M phase cell cycle arrest and cell death, especially apoptosis. These changes may be related to ROS formation, which then cause expressional variations of many genes, such as cdc2, cyclin B, cdc25C, p21, Bax as well as Bcl2. As for HO-1, the induction of its expression may play a role in cell protection, and this is worthy of further study to clarify. Although the concentration of CQ in resin phase is not very high, the concentration of CQ eluate can be high enough to damage pulp cells once the residual CQ diffuses to pulp chamber which has a relatively small volume. Therefore, we should use base materials timely for pulp protection during restoring caries with resin composites in clinical situations. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T04:20:20Z (GMT). No. of bitstreams: 1 ntu-100-R97422004-1.pdf: 2428994 bytes, checksum: 0954ef4f6c472a5b8a358949b0c09bf7 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 中文摘要...ii
Abstract...iv Chapter I. Literature Review...1 1.1. Introduction...1 1.2. Camphorquinone...2 1.2.1. Basic information...2 1.2.2. The visible light activated free radical generation pathway...2 1.2.3. Cytotoxic effects...3 1.3. Cell cycle progression and checkpoints...5 1.3.1. Cell cycle...5 1.3.2. Cell cycle progression...5 1.3.2.1. Cyclin-dependent kinases (CDK) regulation...6 1.3.2.2. Cell cycle inhibitory proteins...6 1.3.2.3. G1/S phase transition...7 1.3.2.4. G2/M phase transition...7 1.3.3. Cell cycle checkpoints...8 1.3.3.1. The G1/S checkpoint...9 1.3.3.2. The intra-S phase checkpoint...10 1.3.3.3. The G2/M checkpoint...10 1.4. Cell death...11 1.4.1. Apoptosis versus necrosis...11 1.4.2. The mechanism of apoptosis...11 1.4.2.1. The caspases...11 1.4.2.2. The Bcl2 family...12 1.5. The role of heme oxygenase-1 (HO-1) in oxidative stress...13 Chapter II. The Purpose of the Study...15 Chapter III. Materials and Methods...16 3.1. Materials...16 3.2. Culture of human dental pulp cells...17 3.3. Morphology of human dental pulp cells...17 3.4. MTT assay...17 3.4.1. Effects of different concentrations of CQ on cell viability...18 3.4.2. Effects of NAC and catalase pre-treatment on cell viability...18 3.4.3. Effects of ZnPP pre-treatment on cell viability...19 3.5. Flow cytometry...20 3.5.1. Cell cycle analysis: PI assay...20 3.5.2. Cell death: PI/Annexin V assay...21 3.5.3. Cellular ROS production: DCF assay...23 3.6. Reverse transcription polymerase chain reaction (RT-PCR)...24 3.6.1. Isolation of total RNA...24 3.6.2. RNA Quantitation...25 3.6.3. Reverse Transcription (RT)...26 3.6.4. Polymerase Chain Reaction (PCR)...26 3.7. Western blot...28 3.7.1. Protein extraction...28 3.7.2. Protein quantification...28 3.7.3. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)...29 3.7.4. Western Blot...30 3.8. Statistical analysis...31 Chapter IV. Results...32 4.1. Density and morphological alterations of human dental pulp cells...32 4.2. Effects of CQ on cell viability of pulp cells: MTT assay...32 4.3. Effects of CQ on cell cycle progression and regulation of pulp cells...32 4.3.1. Cell cycle analysis with PI assay...32 4.3.2. Expression of cell cycle-related genes: focused on G2/M phase...33 4.3.3. Expression of cell cycle-related proteins: focused on G2/M phase...34 4.4. Effects of CQ on cell death of human dental pulp cells...34 4.4.1. PI/Annexin V assay...34 4.4.2. Expression of apoptosis-related genes: focused on Bcl2 family...35 4.4.3. Expression of apoptosis-related proteins: focused on Bcl2 family...35 4.5. Effects of CQ on oxidative stress of human dental pulp cells...36 4.5.1. ROS production: DCF assay...36 4.5.2. Effects of NAC and catalase pre-treatment on cell proliferation...36 4.5.3. Expression of oxidative stress-related genes: focused on HO-1...37 4.5.4. Effects of ZnPP pre-treatment on cell proliferation...37 Chapter V. Discussion...38 5.1. Morphological and proliferation aberrations...38 5.2. Cell cycle aberrations...39 5.3. CQ-induced cell death...43 5.4. CQ-induced oxidative stress to pulp cells...45 Chapter VI. Conclusion...49 References...51 Table 1: Sequences and base pairs of PCR primers...56 Table 2: Protocols of western blot protein extraction buffer...57 Table 3a: Protocol of resolving gel for SDS-PAGE...58 Table 3b: Protocol of stacking gel for SDS-PAGE...58 Table 4a: Protocol of western blot SDS-PAGE running buffer...59 Table 4b: Protocol of western blot transfer buffer...59 Table 4c: Protocol of western blot Tween TBS...59 Table 5: Molecular weight, host and concentration of western blot primary antibodies...60 Table 6: Original data and % of control for MTT assay...61 Table 7: Original data and % of control for DCF assay...62 Table 8: Original data and % of control for MTT assay—effects of NAC or catalase pre-treatment...63 Table 9: Original data and % of control for MTT assay—effects of ZnPP pre-treatment...64 Figure 1a: Structure of camphorquinone...65 Figure 1b: Visible light activated free radical generation pathway...65 Figure 2: Cell cycle...66 Figure 3: A scheme of the cyclin/CDK complexes and inhibitory proteins that regulate cell cycle progression...67 Figure 4: G1/S phase transition and checkpoint...68 Figure 5: G2/M phase transition and checkpoint...69 Figure 6: Mammalian cell cycle checkpoints...70 Figure 7: The Bcl2 family and apoptosis...71 Figure 8: Density and morphology of human dental pulp cells following exposure to CQ for 24 hours...72 Figure 9: Histogram of MTT assay...73 Figure 10a: One representative PI flow cytometry profiles of pulp cells...74 Figure 10b: Quantitative histogram representing the percentage of cells in each cell cycle phase...74 Figure 10c: Quantitative histogram representing the percentage of cells in sub-G0/G1 phase...75 Figure 11: Effects of CQ on mRNA expression of cell cycle-related genes in humandental pulp cells...76 Figure 12: Effects of CQ on the expression of cell cycle-related proteins in human dental pulp cells...77 Figure 13a: One representative PI/Annexin V flow cytometry profiles of pulp cells...78 Figure 13b: Quantitative histogram of PI/Annexin V assay...78 Figure 14: Effects of CQ on mRNA expression of apoptosis-related genes in human dental pulp cells...80 Figure 15: Effects of CQ on the expression of apoptosis-related proteins in human dental pulp cells...81 Figure 16a: One representative DCF flow cytometry profiles of pulp cells...82 Figure 16b: Quantitative histogram of DCF assay...82 Figure 16c: Histogram of MTT assay—effects of NAC or catalase pre-treatment...83 Figure 17a: Effects of CQ on mRNA expression of HO-1 in human dental pulp cells...84 Figure 17b: Histogram of MTT assay—effects of ZnPP pre-treatment...84 | |
dc.language.iso | en | |
dc.title | 樟腦醌對人類牙髓細胞之細胞毒性,細胞週期分佈以及細胞凋亡相關基因與蛋白表現的影響 | zh_TW |
dc.title | Effects of camphorquinone on cytotoxicity, cell cycle regulation, apoptosis related gene and protein expression to human dental pulp cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 許明倫,涂明君,張美姬 | |
dc.subject.keyword | 人類牙髓細胞,樟腦醌,細胞毒性,細胞週期停滯,細胞凋亡, | zh_TW |
dc.subject.keyword | apoptosis,camphorquinone,cell cycle arrest,cytotoxicity,dental pulp cells, | en |
dc.relation.page | 84 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-07-28 | |
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 | |
---|---|---|---|
ntu-100-1.pdf 目前未授權公開取用 | 2.37 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。