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

Effects of camphorquinone on cytotoxicity, cell cycle regulation, apoptosis related gene and protein expression to human dental pulp cells

吳敏慈(Min-Tsz Wu)

指導教授 ： 鄭景暉

國立臺灣大學/牙醫專業學院/臨床牙醫學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02949

摘要

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。

關鍵字

人類牙髓細胞 ； 樟腦醌細胞毒性 ； 細胞週期停滯 ； 細胞凋亡

並列摘要

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.

並列關鍵字

apoptosis ； camphorquinone ； cell cycle arrest ； cytotoxicity ； dental pulp cells

參考文獻

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.