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研究生:呂佳珮
研究生(外文):Chia-PeiLu
論文名稱:含氟漂白劑對牙髓細胞的毒性影響
論文名稱(外文):Cytotoxic effects of fluoridated bleaching gel on human dental pulp cells
指導教授:莊淑芬莊淑芬引用關係陳玉玲陳玉玲引用關係
指導教授(外文):Shu-Fen ChuangShu-Fen Chuang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:口腔醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:44
中文關鍵詞:漂白劑牙髓細胞細胞毒性分化礦化
外文關鍵詞:bleaching agentsfluoridedental pulp cellscytotoxicitydifferentiationmineralization
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牙齒美白是牙科美學之重要項目。診間與居家美白是常用於牙齒漂白的技術,主要利用不同濃度的過氧化氫(H2O2)或碳醯胺過氧化物(carbamide peroxide, CP)的漂白劑進行美白。漂白的原理是利用氫氧基能穿透牙體結構並且產生自由基以氧化色素分子,但會伴隨牙齒去礦化和相關過敏反應的發生。之前的研究證明含氟漂白劑藉由降低氧化劑滲透的含量或是增加琺瑯質的礦化來預防副作用。然而,這些含氟漂白劑需要更多方面的評估,尤其是對牙髓細胞生物毒性。本研究提出評估含氟漂白劑對牙髓細胞反應的影響。
第一部分,實驗組別分為五組,第一組沒有處理漂白劑 (控制組),第二組使用含10%不含氟的碳醯胺過氧化物,第三組是處理10%含0.11%的氟化物以及第四組為15%含0.11%氟化物之碳醯胺過氧化物作用於人工牙髓腔作用1、2、4、8小時,另取第五組35%含0.11%氟化物之漂白劑作用30分鐘,作用於人工牙髓腔 (體積為400μL)。測量含氟漂白劑在不同臨床情況下,滲透牙釉質-牙本質及牙本質過氧化氫的含量。過氧化氫滲透含量的濃度利用HRP/OPD 方法分析,結果顯示作用於牙釉質-牙本質或牙本質8小時後滲透量與濃度與置放時間呈正相關。第五組作用於牙本質30分鐘後已可測出滲入量0.28 mM。第二部分,評估含氟漂白劑對人類牙髓細胞的細胞毒性。細胞存活率由WST-1進行評估分析,細胞毒性隨著過氧化氫濃度隨著濃度增高 (0-1 mM) 而增加,而在滲入的氟化物濃度 (0-5 ppm) 並不會造成顯著的細胞毒性。過氧化氫 (0.4、0.6、0.8 mM) 和1 ppm的氟化物一起共同培養亦不會增加過氧化氫所造成的細胞毒性。第三部分,評估含氟漂白劑對人類牙髓細胞的分化、礦化功能的影響。由結果發現使用0.5 mM的過氧化氫會減少牙本質母細胞相關基因、蛋白質的表現量以及減少鹼性磷酸酶活性 (ALP activity) 和礦物質小結的形成。然而,如果將0.5 mM的過氧化氫和1 ppm的氟化物一起共同培養,會促進牙髓細胞礦化。研究結果顯示過氧化氫的濃度提高會影響細胞毒性與細胞分化。含氟的漂白劑可減少過氧化氫滲透的含量,但對氟化物滲透入牙髓腔並無明顯影響。微量的滲入氟化物,也不會對細胞表現有所影響。使用含氟的漂白劑伴隨著微量的氟化物滲入,可促進牙髓細胞礦化。

Tooth bleaching is a common treatment for correcting dental esthetics. For both in-office and at-home bleaching, the primary reactive agents are hydrogen peroxide and carbamide peroxide (CP). The bleaching efficacy is based on the ability of release peroxide to penetrate the tooth structure and produce free radicals that oxidize the coloured organic molecules. During the procedures, some side effects including the tooth demineralization and sensitivity are noted. Previous studies have suggested that fluoridated bleaching agents may prevent these associated problems. However, these fluoridated bleaching agents need more evaluation on the biological aspect of their cytotoxic effects especially on pulp cells. This study was proposed to evaluate effects of fluoridated bleaching agents on the cell responses of the pulp cell.
The first experiment was to measure the diffusion of H2O2 into dental pulp during bleaching. The following four groups: Group 1, untreated controls; Group 2, treatment with 10% carbamide peroxide (CP) bleaching agent; Group 3, treatment with 10% CP containing 0.11% fluoride; Group 4, treatment with 15% CP and 0.11% fluoride. The four groups were applied on the artificial pulp chamber for 1, 2, 4, 8 h to measure the dose of hydrogen peroxide penetrating though enamel-dentin or sole dentin. Additionally, 35% CP containing 0.11% fluoride (group 5) was also applied for 30 min to simulate the in-office bleaching. The trans-enamel and dentine penetration was analyzed by HRP/OPD assay for determination of the H2O2 concentration. For sole dentin, the CP groups showed time-dependent of H2O perfusion. After 8h, G2 showed a higher concentration of H2O2 penetration (1.51 mM) than that of G3 (1.28 mM). The application of G5 for 30 min resulted in 0.28 mM of H2O2 penetration. The second experiment was to measure the cytotoxicity of fluoridated bleaching agents were evaluated on human dental pulp cells. Cell viability was evaluated by the WST-1 assay. The results showed that H2O2 induced a concentration-dependent (0-1 mM) cytotoxic effect and decreased odontoblast-related gene and protein expression treated pulp cells, while fluoride in the diffusion (0-5 ppm) was not significantly cytotoxic. Moreover, co-incubation with 1ppm fluoride unable to enhanced the cytotoxic effects of H2O2 (0.4, 0.6, 0.8 mM) on pulp cells. The third experiment was to measure the differentiation, and mineralization functions of fluoridated bleaching agents were evaluated on human dental pulp cells. H2O2 at concentration of 0.5 mM was decreased the mRNA levels, protein levels of differentiation markers, ALP activity and mineralized nododules formation of pulpc ells. Moreover, our data also indicate that 1 ppm fluoride, when co-incubated with 0.5 mM H2O2, can promote mineralization of dental pulp cells. The fluoridated bleaching agents can reduce the penetration of active bleaching agent into the tooth structure.

中文摘要 I
Abstract III
Acknowledgement V
Contents VII
List of Tables IX
List of Figures X
Abbreviation XI
Introduction 1
1.Tooth bleaching 1
2.Bleaching mechanisms and side effects with tooth
bleaching 1
3.Fluoridated bleaching gel and mechanism 3
4.Toxicity of fluoride on cell 4
5.Human dental pulp cells (HDPCs) 4
Motivation and Specific Aims 6
Materials and Methods 7
Reagents and Equipments 7
1.Primary culture of human dental pulp cells 8
2.Trans-enamel and dentin diffusion of bleaching agents 8
3.Chemical analysis of diffusion from bleaching agents 10
4.Measurement of fluoride 11
5.WST-1 assay 11
6.Cell apoptosis 12
7.Cell morphology 12
8.RNA Isolation and Quantitative Real-time PCR (qRT-PCR)
analysis 13
9.Western Blot Analysis 14
10.ALP Activity 15
11.Alizarin red S stating 15
Results 17
1.H2O2 penetrated through sole dentin and enamel-dentin in a time-dependent manner 17
2.Cytotoxicity of H2O2 on dental pulp cells 19
3.Induction of apoptosis of dental pulp cells by H2O2 21
4.Cytotoxicity of fluoride on dental pulp cells 22
5.H2O2 induced morphologic changes of pulp cells 24
6.Effects of H2O2 on differentiation of dental pulp cells 25
7.Effects of H2O2 on ALP activity and mineralization of dental pulp cells 26
Discussion 29
Conclusion 35
Reference 36
Appendix 42
Appendix1. Reagents 42
Appendix2. Equipments 44

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