甲基丙烯酸羥乙酯單體的毒性研究與分析

Abstract

中文摘要 聚合黏著系統及光聚合樹脂大量的使用於臨床工作之中，但常發現有些病人在使用這些材料填補齲齒後，發生術後敏感甚至是牙髓發炎的現象。黏著系統中所使用的單體是否具有毒性，長久以來，已是備受爭議的問題。一般認為樹脂單體在到達一定濃度後具有細胞毒性，但是這類單體對細胞影響的作用機轉尚未曾明朗。 本實驗目的在於研究牙科黏著系統中最常見的甲基丙烯酸羥基乙酯（2-Hydroxyethyl Methacrylate，HEMA）對於人類嬰兒牙髓細胞(Human baby dental pulp cell，HDPC)及小鼠胸腺纖維母細胞株(NIH/3T3)所造成的影響。本實驗測試使用半數中毒濃度(median toxic concentration，TC50)以下單體濃度培養細胞後，細胞在短期(24hr)、中期(72hr)，長期(120hr)的細胞反應。首先先以細胞存活率分析((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay)測試HEMA對兩種細胞在24小時，72小時，以及120小時的TC50，分別約為HDPC:24小時10mM、72小時4mM 、120小時2.4mM；以及3T3 24小時7.4mM、72小時3.2mM、120小時2.67mM。 之後以0mM(未加單體)、0.25mM、1mM、2.5mM、5mM，以及10mM濃度培養上述兩種細胞24小時後，觀察細胞存活狀況；中期(72hr)以及長期(120hr) 的培養濃度分別為0mM、0.125mM、0.25mM、1mM、2mM、4mM。同時，在光學顯微鏡下觀察HEMA單體對細胞形態結構上所造成的改變。本實驗更進一步利用流式細胞儀(flow cytometry)以及Annexin V/PI雙染法測量經過HEMA單體處理後，HDPC以及3T3所引起的細胞死亡模式。之後，為了針對臨床治療效果，進行更深入的研究，我們將實驗集中於人類嬰兒牙髓細胞。 本實驗為了探討HEMA對人類牙髓細胞穀胱甘肽(Glutathione,GSH)系統的影響，利用測定GSSG/GSH的方式來檢驗細胞中的GSH是否真的被HEMA單體所產生的氧化壓力(oxidative stress)消耗。結果證實，對照控制組，接觸HEMA濃度越高則GSH消耗的越多。另外為了解其觸發的信息路徑，也利用免疫酵素連結測試(ELISA)測定磷酸化的NF-ĸB(phosphor NF-ĸB)與全體NF-ĸB(total NF-ĸB)，以確認NF-ĸB是否在HEMA單體觸發牙髓細胞進入凋亡的信息傳遞中扮演重要的角色；實驗中發現，在短期（24hr，72hr）接觸的情況下，磷酸化NF-ĸB的量與細胞凋亡的比率成現相反的趨勢意味著NF-ĸB可能能夠保護細胞避免凋亡，而在五天的實驗觀察發現磷酸化NF-ĸB隨著細胞凋亡比率上升而上升，或許意味著NF-ĸB在長期接觸下導致細胞走向凋亡。

關鍵詞： 甲基丙烯酸羥基乙酯、牙髓細胞、毒性、黏著系統、單體、穀胱甘肽

Abstract Dentin bonding system (DBS) was first introduced about over 50 years ago. Nowadays, it becomes more and more popular and necessary in dental practice. It provides aesthetic and strong bonding strength between tooth structure and restorations. Some studies revealed the chemical component in DBS such as monomers may irritate the pulp complex lead to pulp in-flammation and post-operative hypersensitivity. Evidences showed chemical monomers at certain concentrations is cytotoxic , and might cause cell apoptosis. Although the detail mechanism is unclear, several hypotheses have been proposed. This study will focus one of the most common monomer used in DBS: 2-Hydroxyethyl Methacrylate (HEMA). Frist, the MTT assay was taken to determine the concentrations of TC50 of NIH/NIH/3T3 cell line and HDPC on HEMA. The results of TC50 of HEMA to HDPC at 24hrs is about 10mM, 72hrs is 4mM, and 120hrs is 2.4mM ; TC50 of HEMA to NIH/3T3 cell is about 7.4mM, 72hrs is 3.2mM, and 120hrs is 2.67mM. Then, the lower concentration (for 24hrs test: 0mM, 0.25mM, 1mM, 2.5mM, 5mM, and 10mM ; for 72hrs and 120 hrs : 0mM, 0.125mM, 0.25mM, 1mM, 2mM, and 4mM) was used in cell culture and analyzed with MTT assay. Also, to observe the changes of cell morphology of HEMA-treated cells, the photos were taken under light microscope 200x. Moreover, in or-der to investigate the pattern of cell death, the FITC-Annexin V/PI Dual staining was carried out on HDPC and NIH/3T3 cells , under all concentration of HEMA monomer which are mentioned before. This study also used fluorescent staining technique to enhance cell skeleton by Phalloidin stain and cell DNA was stained by using Hoechst stains in order to observe cell morphology changing and apoptotic bodies. And then we focus on HPDC cell use GSSG/GSH ELISA test and NF-ĸB p65 (to-tal/phosph) assay hopefully to obtain further information of HEMA induced ROS increases and GSH depletion and activate NF-ĸB signal pathway. The initial result indicated that in HEMA induced cell apoptosis, cell apoptosis rate rise with GSH depletion, and for phosphor NF-ĸB, in short term (<72hr), NF-ĸB may protect cell from HEMA induced apoptosis; in long term (120hr), NF-ĸB may lead cell to apoptosis in-duced by HEMA.

Key words: monomer, HEMA, HDPC, cytotoxicity, GSH, NF-κB