"UDMA誘導人類牙髓細胞產生氧化壓力,影響細胞週期進行及發生細胞凋零之效應"

Abstract

實驗目的： 雙甲基丙烯酸氨基甲酸乙酯（UDMA），是牙科樹脂材料中重要且現今常被使用的單體之一，未聚合的單體釋放是造成組織細胞毒性的來源。本研究的目的在於探討UDMA對人類牙髓細胞的毒性，以及對產生氧化壓力(ROS),影響細胞週期進行及發生細胞凋零(apoptosis)之效應做進一步的探討。 實驗方法： 將人類牙髓細胞暴露於不同濃度的UDMA(0.025~0.35mM)下培養後，以顯微鏡觀察細胞的形態以及數量變化，以MTT assay來測定細胞存活的比例，另以流式細胞儀（flow cytometry）來進行細胞週期的分析。藉由反轉錄聚合酶連鎖反應(RT-PCR)和西方墨點法(Western blot)來觀察基因以及蛋白的表現。也分析活性氧過高(ROS overproduction)與氧化壓力相關基因(HO-1, CES2)和發炎相關(COX-2)基因及蛋白表現之間的關係。至於UDMA所導致產生的活性氧與各類抗氧化劑之間的關係，是藉由加入抗氧化劑NAC (1, 3 mM) 和catalase (1000, 2000 U/ml)、esterase (2, 4 U/ml) 前處理30分鐘再暴露於0.35 mM UDMA濃度下作用24小時後做MTT assay來證實。另加入HO抑制劑Zn-P (1, 2.5 μM)、 CES抑制劑BNPP (0.5, 1 mM)和CES2抑制劑 Loperamide (10, 20 μM)前處理30分鐘的牙髓細胞緊接著暴露於UDMA 0.1mM 濃度下共同培養24小時後再以MTT assay測定細胞存活率的變化。統計方式是使用單因子變異數分析搭配Tukey事後檢定來分析實驗數據。 實驗結果： 人類牙髓細胞經濃度0.25 mM和0.35 mM UDMA作用24 小時之後，可觀察到細胞形態之改變和細胞存活率降低的情況。在MTT assay結果中可見，0.1mM UDMA能造成細胞存活率下降至約70%，而加入0.25 mM和0.35 mM UDMA作用後，細胞存活率更是下降至約40%。在0.35 mM UDMA濃度下能造成細胞週期的分布變化，G0/G1細胞數量的下降達統計上的顯著差異，G2/M細胞數量有明顯上升，但未達統計上的顯著差異（P=0.32），同時相關基因和蛋白cdc2, cyclin B1, cdc25C的表現被抑制，p21則是有基因表現上的些微促進。在細胞凋零相關的基因和蛋白表現中，Bax、Bad被促進，Bcl2被抑制。在加入超過0.1mM的UDMA作用24小時候可以看到HO-1表現量上升, 在加入超過0.25 mM的UDMA作用24小時候，CES2和COX-2的表現量才大幅上升。實驗也發現，經過catalase、esterase和高濃度NAC前處理的細胞，UDMA所造成的細胞存活率降低現象會被抑制，而Zn-P、BNPP和Loperamide前處理的細胞則是會更加促進UDMA所造成的細胞毒性。 結論： UDMA超過0.1 mM就會造成一定程度的細胞毒性以及活性氧的顯著增加，而UDMA濃度高過0.25 mM則會有非常明顯的細胞毒性。UDMA在0.35mM的濃度下會造成細胞週期G0/G1的細胞數量減少。這些細胞型態改變、存活率降低、細胞週期停滯都是與活性氧產生有關，進一步造成細胞週期相關基因及蛋白(cdc2, cyclin B1, cdc25C抑制，p21些微促進)的表現變化，以及細胞凋零相關基因和蛋白(Bax、Bad被促進，Bcl2被抑制)表現的變化。另外，HO-1和CES2可能扮演了保護細胞的角色，對於細胞遭受氧化壓力做出反應。COX-2的基因以及蛋白表現則證實UDMA與牙髓細胞所產生的慢性發炎反應有關，需進一步再去研究證實。另外在抗氧化劑以及氧化壓力相關基因的抑制劑共同作用下則會影響UDMA所導致的細胞毒性，其可能與阻止或增強活性氧的產生有關。雖然在牙科樹酯產品中的UDMA濃度並不會到達很高的濃度，但當未聚合的UDMA擴散到體積相對較小的牙髓腔時，所滲出的濃度所造成的毒性就有可能相對較高，進而對牙髓細胞產生相當的毒性。對體外細胞培養來做UDMA毒性的探討將有助於牙醫師在臨床應用上，使用上須慎選樹酯的使用條件以及評估合適的臨床狀況，適時使用基底材料保護牙髓，降低病人在填補後所產生的牙髓慢性發炎甚至壞死的不良反應。

Aim: Urethane dimethacrylate (UDMA) is one of the monomers usually be included in dental resin materials. The purpose of our study is to investigate the effects of UDMA on cytotoxicity to human dental pulp cells, involving the relationship between ROS formation and UDMA toxicity. The influences UDMA on the expression of cell cycle- and apoptosis-related genes and proteins were also evaluated. Materials and methods: Primary-cultured human dental pulp cells were obtained from human extracted premolars and third molars. Cells were treated with different concentrations of UDMA (from 0.025 to 0.35 mM), incubated 24 hours and then observed the changes of cell morphology in phase contrast microscope. Cell proliferation was evaluated by MTT assay. Cell cycle analysis was investigated by flow cytometry. Influences in mRNA expression were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and the changes in protein production were determined by Western blot. For the purpose of link the rule of ROS and antioxidant mechanism, pulp cells were pre-treated for 30 minutes with NAC (1, 3mM) and catalase (1000, 2000 U/ml), esterase (2, 4 U/ml) to check their ability of removing overproduced ROS. And Nrf2, HO-1, CES1A1, CES2 and CES3 mRNA expression can be the evidences of cells facing oxidative stress. By adding HO inhibitor Zn-P (1, 2.5 μM), CES inhibitor BNPP (0.5, 1 mM) and CES2 inhibitor Loperamide (10, 20 μM) 30 minutes before co-incubation with 0.1mM UDMA to evaluate the influences to cell viability by MTT assay. And the testing of expression of COX-2 gene and protein was to link UDMA and chronic inflammatory effect. One-way ANOVA and post hoc Tukey test were used to analyze differences between experimental and control groups. Results: In human dental pulp cells, UDMA induced morphological changes and a significant decrease of cell viability at the concentration of 0.1mM UDMA to about 70%, and at the concentration of 0.25 and 0.35 mM UDMA to about 40%. At the concentrations of 0.35mM UDMA, it could lead to increase of G2/M cell percentage and decrease of G0/G1 cell percentage. The expression of production of cdc2, cyclinB1, cdc25C was inhibited, while that of p21 was slightly promoted in both PCR and Western blot examination. For apoptosis analysis, Bax, Bad were promoted, and Bcl2 was inhibited. Besides, the increase of HO-1, CES2 and COX-2 expression was noted after 24 hours. The reduction of cell viability caused by UDMA can be inhibited by NAC, catalase and esterase pre-treatment, and can be promoted by BNPP, loperamide and Zn-P pre-treatment. Conclusion: UDMA at a concentration higher than 0.1 mM had a significant cytotoxicity. Under the concentration higher than 0.1 mM, UDMA can cause changes of cell morphology, reduction of cell viability, and 0.35mM UDMA can cause increase of G2/M phase cell percentage. These changes may be related to ROS overproduction, which can cause expressional variations of many genes, such as cdc2, cyclin B1, cdc25C, p21, Bax as well as Bcl2. As for HO-1 and CES2, the induction of its expression may play a role in cell protection. The finding of COX-2 gene and protein expression suggest UDMA can be a critical factor of inflammation related to resin-based dental biomaterials, and that COX-2 is involved in the inflammatory reaction of the resin monomers. We know the concentration of UDMA in dental resin product is not very high, but the concentration of UDMA elute can be high enough to damage pulp cells once the residual UDMA diffuses to pulp chamber with a relatively small volume. Therefore, we should use resin materials carefully in daily practice and should select proper case and the situation of enough residual dentin thickness to do the resin restoration procedure.