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

• Main Authors: Min-Tsz Wu, 吳敏慈
• Other Authors: 鄭景暉
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/40821330045076065750

碩士 === 國立臺灣大學 === 臨床牙醫學研究所 === 99 === 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.