雙甲基丙烯酸三縮乙二酯對牙髓細胞的細胞週期調控、Nrf2/HO-1、NQO1及Cox-2的表現:活性氧和TGF-β活化激酶1之角色

Effect of TEGDMA on cell cycle regulation, Nrf2/HO-1, NQO1 and COX-2 expression on dental pulp cells: Role of reactive oxygen species and TAK1

蕭美美(Mui-Mui Sue)

指導教授 ： 鄭景暉教授

共同指導教授 ： 張曉華助理教授

國立臺灣大學/醫學院/臨床牙醫學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702035

摘要

目的: 雙甲基丙烯酸三縮乙二酯為目前廣泛地在牙科樹脂材料中使用。本研究的目的著重在瞭解雙甲基丙烯酸三縮乙二酯在人類牙髓細胞中，藉由自由基的累積而造成的氧化壓力所引起的細胞毒性以及刺激細胞的發炎反應；此外，在氧化壓力下所啟動的Nrf2訊息傳遞，其所轉譯蛋白的表現。最後，我們也希望進一步探討人類牙髓細胞在氧化壓力下，自由基以及TGF-β活化激酶1所扮演的角色。

實驗方法：將人類牙髓細胞取出培養，將其暴露於不同濃度（0.35-0.7-1.75-3.5mM）的雙甲基丙烯酸三縮乙二酯下24小時，以反轉錄聚合酶連鎖反應（RT-PCR）及西方點墨法（Western blot）來評估調控細胞週期(如:cdc2、cyclinB1、cdc25C及p21)及細胞發炎反應表現(COX-2)的基因及蛋白質。此外也針對當細胞在雙甲基丙烯酸三縮乙二酯作用時，引起自由基的產生，讓牙髓細胞處在氧化壓力下，Nrf2及其下游轉譯基因及蛋白的表現。另外也會透過加入自由基清除劑－過氧化氫酶以及TAK1 抑制劑－(5Z)-7-oxozeaenol，來探討活性氧以及TGF-β活化激酶1訊息傳遞路徑在雙甲基丙烯酸三縮乙二酯所誘導的毒性下的角色。最後，藉由免疫螢光染色來觀察調控細胞週期的上游蛋白p-ATM的表現。

實驗結果：人類牙髓細胞暴露在雙甲基丙烯酸三縮乙二酯24小時後，在濃度1.75mM時，cyclinB1、cdc2及cdc25C的表現是下降,而p-21的表現是增加的，表示雙甲基丙烯酸三縮乙二酯對細胞在高濃度時，會對細胞週期都有所影響。隨著雙甲基丙烯酸三縮乙二酯濃度作用在人類牙髓細胞的增加，在濃度1.75mM及3.5mM 時，COX-2的表現是增加的，細胞在雙甲基丙烯酸三縮乙二酯會誘發發炎反應的發生。在經過雙甲基丙烯酸三縮乙二酯作用下,人類牙髓細胞內會形成活性氧，並且累積而造成細胞內的氧化壓力，Nrf2及其轉譯的抗氧化酶之基因及蛋白質在這樣的狀態下，其表現也是增加的，表示Nrf2在氧化壓力下,會被活化並且啟動其下游轉譯的抗氧化酶。在加入濃度500及 1000 U/ml過氧化氫酶的條件下，調控細胞週期的cdc2的表現有被增強，p21被抑制下來；而被氧化壓力所活化的Nrf2及其轉譯的抗氧化酶的表現也都有被抑制的現象，表示細胞因氧化壓力所引起的細胞週期停滯、Nrf2的活化和自由基的堆積是有相關性的。另外，在加入2.5、5µM的(5Z)-7-oxozeaenol作用後，發現細胞週期的調控並沒有被TAK1抑制劑所影響，而COX-2及Nrf2均有被TAK1抑制劑所抑制；不同的是，抗氧化蛋白酶如HO-1的表現和Nrf2是相反的。TGF-β活化激酶1可以對細胞發炎的表現以及Nrf2有所影響，或許細胞週期的調控是被TGF-β活化激酶1以外的訊息傳遞路徑所影響的；HO-1有可能在細胞面對氧化壓力時，被更強勢的訊息傳遞所控制，而有表現增加的結果。最後，從免疫螢光染色的結果，亦可看出 p-ATM在人類牙髓細胞中分布的表現，隨著濃度的增加，p-ATM亦有增加的表現。

結論：臨床上，當雙甲基丙烯酸三縮乙二酯在人類牙髓組織中，會對牙髓造成影響而導致牙髓發炎或是牙髓壞死的可能。雙甲基丙烯酸三縮乙二酯會引起活性氧的堆積及而形成氧化壓力的狀態，對細胞造成遺傳毒性且影響細胞週期的調控，同時也會誘發細胞發炎反應以及抗氧化路徑 - Nrf2及其下游的抗氧化酶來應付細胞內的氧化還原的平衡。TAK1訊息傳遞對於雙甲基丙烯酸三縮乙二酯所引起的COX-2, Nrf2及NQO1都是有影響的。

關鍵字

雙甲基丙烯酸三縮乙二酯 ； 人類牙髓細胞 ； 細胞週期 ； 抗氧化酶 ； 過氧化氫酶 ； TGF-β活化激酶1

並列摘要

Aim: Triethylene glycol dimethacrylate (TEGDMA) is one of the most widely used resin monomer in dental resin-based materials. TEGDMA has been reported to induce accumulation of reactive oxygen species and cytotoxicity in human dental pulp cells (HDPCs). The aim of this study is to investigate the effect of TEGDMA on cell cycle regulation, COX-2 production, as well as expression of oxidative stress responsive genes Nrf2, NQO1 and HO-1 in HDPCs. The role of antioxidant and TAK-1 signaling were also evaluated.

Materials and methods: Primary-cultured HDPCs were treated with different concentrations (0.35-0.7-1.75-3.5mM) of TEGDMA for 24 hours. The expression of cell cycle related factors (eg. cdc2, cyclinB1, cdc25C, and p21) and the proinflammatory cyclooxygenase, COX-2 were evaluated through RT-PCR or western blot. The Nrf2 and the downstream antioxidant response element-regulated targets (eg. NQO1, HO-1) were also evaluated. In addition, pretreatment of catalase (as the ROS scavenger) and (5Z)-7-oxozeaenol (as a specific TAK1 inhibitor) were used to investigated the roles of ROS and TAK1 signaling in TEGMDA-induced effect on HDPCs. At last, the expression and distribution of upstream cell cycle checkpoint component p-ATM were evaluated through immunofluorescence.

Results: HDPCs exposed to TEGDMA for 24 hours showed alteration of cell cycle related factor at the concentration 1.75, 3.5mM. TEGDMA suppressed cyclinB1, cdc2, and cdc25C while induced p21 expression. COX-2 was upregulated at 1.75-3.5mM of TEGDMA which may represented the pro-inflammatory response. The expression of genes and proteins of Nrf2, NQO1 and HO-1 were increased when HDPCs exposed to TEGDMA. Pretreatment of 500 and 1000 U/ml of catalase reduced the TEGDMA-induced expression of Nrf2 and HO-1. By adding of 2.5 and 5µM (5Z)-7-oxozeaenol, the TEGDMA-induced expression of COX-2, Nrf2 and NQO1 was inhibited. The analysis of immunofluorescence showed p-ATM was enhanced when the concentration of TEGDMA increased.

Conclusions: TEGDMA may induce adverse response to HDPCs which can be related to cell cycle disturbance and pro-inflammatory effect. The oxidative stress responsive genes including Nrf2, NQO1, and HO-1 were upregulated to provide cytoprotective function. Administration of antioxidant may reduce TEGDMA-induced oxidative stress thus lessen the expression of Nrf2 and HO-1. TAK1 signaling may convey signal transduction for TEGDMA-induced COX-2, Nrf2 and NQO1 expression.

並列關鍵字

TEGDMA ； human dental pulp cells ； cell cycle ； Nrf2 ； antioxidant-response element ； catalase ； TAK-1

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