甲基丙烯酸 2-羥乙酯對牙髓細胞之毒性機制及其預防方法探討

Toxic Effect of HEMA (2-hydroxyethyl methacrylate) on Human Dental Pulp Cells and Its Prevention

陳郁仁(Yu-Jen Chen)

指導教授 ： 鄭景暉

共同指導教授 ： 張曉華

國立臺灣大學/醫學院/臨床牙醫學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902532

摘要

目的:甲基丙烯酸 2-羥乙酯為牙本質黏著劑及牙科複合樹脂內重要成分，且相關 材料於牙科醫療被廣泛使用。然而，甲基丙烯酸 2-羥乙酯亦為常見釋出單體種 類。這類釋出樹脂單體可能影響牙髓組織細胞活性或導致各種不良生物效應，但 目前仍尚未完全瞭解其中相關之毒性及作用機制。本研究目的著重於探討甲基丙 烯酸 2-羥乙酯對於人類牙髓細胞中的細胞基質、細胞週期調節分子、抗活性氧化 物分子和發炎分子之訊息傳遞及其轉譯蛋白表現。此外，實驗更進一步透過加入 抗氧化劑乙醯半胱氨酸( N-Acetyl Cysteine )與褪黑激素，探討相關作用機制是否 受改變;希望藉此找尋幫助細胞適應樹脂單體毒性的方法。
實驗方法:將人類牙髓細胞取出培養，並暴露於不同濃度的甲基丙烯酸 2-羥乙酯 (1, 2.5, 5, 7.5, 10 mM)下 24 小時後，以 MTT 測定評估細胞存活，並以反轉錄 聚合酶連鎖反應(RT-PCR)及西方點墨法(Western blot)來評估細胞基質、調 控細胞週期、細胞氧化壓力和細胞發炎反應表現的基因及蛋白質表現之改變。實 驗第二部分會將人類牙髓細胞先培養於特定濃度的抗氧化劑乙醯半胱氨酸或褪黑 激素一小時後，再加入甲基丙烯酸 2-羥乙酯(10 mM)培養 24 小時，並觀察細 胞相關作用機制是否改變。
實驗結果:人類牙髓細胞經濃度 7.5 mM 和 10 mM 的甲基丙烯酸 2 -羥乙酯作 用可觀察到細胞存活率降低的情形。而基因與蛋白質表現的實驗結果顯示 24 小 時的甲基丙烯酸 2 -羥乙酯暴露，會促使人類牙髓細胞的 Collagen typeI和 cdc2 表現下降，p-21、NQO1、Nrf2、HO-1 及 COX-2 的表現會上升;然而，SOD-1 表現並沒有受到單體暴露影響。實驗第二部份結果可以觀察到，原先由甲基丙烯 酸 2-羥乙酯乙醯引起的 Collagen typeI表現下降，會因加入乙醯半胱氨酸與褪黑 激素而些微回升。然而針對相關細胞週期調節分子、發炎分子以及受氧化壓力調 節之 Nrf2 與 HO-1 則會因加入抗氧化劑之不同，而有不同改變。
結論:本實驗使我們得以洞悉甲基丙烯酸 2-羥乙酯對人類牙髓細胞產生毒性的機 制。其中細胞基質更新與細胞週期的損傷、細胞發炎反應、誘發 Nrf2、HO-1 及 NQO-1 的表現，都可能是臨床使用牙本質黏著劑和複合樹脂時，人類牙髓細胞在 面對甲基丙烯酸 2-羥乙酯暴露的反應方式。而抗氧化劑乙醯半胱氨酸與褪黑激素 均可能透過回復細胞基質更新來幫助細胞面對甲基丙烯酸 2-羥乙酯暴露。但其他 蛋白表現顯示兩者就細胞週期調節、抗氧化及抗發炎之作用可能為不同機制。

關鍵字

甲基丙烯酸 2 -羥乙酯 ； 人類牙髓細胞 ； 乙醯半胱氨酸 ； 褪黑激素 ； 細胞週期 ； 氧化壓力 ； 發炎反應

並列摘要

Aim: HEMA (2-hydroxyethyl methacrylate), one of the main components of dentin bonding agent and dental composite resin, may potentially affect the viability and biological activities of the dental pulp. However, the underlying mechanisms remain largely unknown. This study aimed to investigate the influences of HEMA on the expression of extracellular matrix protein (type I collagen), cell cycle related molecules (p21, cdc2, cdc25c, cyclin B1), Nrf2, Nrf2-related drug-metabolizing proteins (HO-1, NQO1, SOD-1), and inflammatory molecule (COX-2). The effect of antioxidant NAC and melatonin on several relevant proteins’ expression were also evaluated.
Materials and methods: Primary human dental pulp cells were treated with different concentrations of HEMA (0-10 mM) for 24 hours. Cell viability was measured by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Changes in cellular mRNA expression were determined by reverse-transcription polymerase chain reaction (RT-PCR). Changes in protein production were evaluated by western blot. In the second part of this study, human dental pulp cells were exposed to 10 mM HEMA for 24 hours, with and without 1 hour pretreatment of antioxidant NAC or melatonin, to investigate the effect of exogenous antioxidant on HEMA-induced cytotoxicity.
Results: HEMA may induce cytotoxic effect by impairing cell viability on human dental pulp cells at higher concentrations. The expressions of collagen type I, and cdc2 in dental pulp cells were down-regulated after exposure to HEMA for 24 hours. The expressions of p21, NQO-1, Nrf2, HO-1, and COX-2 showed up-regulation, whereas SOD-1 showed no marked change by monomer exposure. Both NAC and melatonin seem to restore the impaired protein expression of collagen type I. However, the effect of NAC and melatonin toward cell cycle, ROS metabolism, and inflammation-related proteins showed different results.
Conclusions: This study provides an insight into the mechanism of HEMA-induced toxicity toward human dental pulp cells. Impairment of matrix turnover and cell cycle progression, as well as induction of Nrf2, HO-1, NQO1, and COX-2 inflammatory response by HEMA, may contribute to the response of dental pulp to dentin bonding agents and composite resin materials after clinical operative procedures. Although it is generally accepted that un-controlled ROS productions contribute to the cytotoxicity and genotoxicity of HEMA, supplement of different antioxidants might protect human dental pulp cells in different ways. Further investigation is needed for finding the successful way to prevent monomer-induced adverse biological effect.

並列關鍵字

HEMA ； Human dental pulp cells ； NAC ； Melatonin ； cell cycle ； oxidative stress ； inflammatory response

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