比較五種傳統與奈米級牙本質黏著劑毒性機轉之探討

Mechanisms of Cytotoxicity by Five Conventional and Nano Dentin Bonding Agents to CHO-K1 cells

葉宏偉(Hung-Wei Yeh)

指導教授 ： 鄭景暉

共同指導教授 ： 林俊彬

國立臺灣大學/醫學院/臨床牙醫學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.01355

摘要

實驗目的: 本實驗是藉由中國倉鼠卵巢細胞的體外試驗，評估並比較市售的五種牙本質黏著劑對細胞毒性及基因毒性的影響，所選擇的廠品包括3種成分為奈米填料(B:Voco Futurabond NR, C: 3M Singlebond 2, D:Dentsply Prime & Bond NT)、2種成分為傳統的非奈米填料(A:Voco Solobond M, E:Dentsply Xeno III)。
實驗材料與方法: 中國倉鼠卵巢細胞在五種牙本質黏著劑以不同濃度(1/40000-1/20, v/v, %)及陰性對照組(0.5%的DSMSO)作用下暴露24小時。細胞毒性的評估是利用細胞群落形成效率分析(CFE)，並分別利用PI (40ug/ml)和DCFH-DA的染色下，利用流式細胞儀進行細胞週期及活性氧的評估。再利用Modfit軟體進行G0/G1期, S期, G2/M期和sub-G0/G1等細胞週期所佔比例的評估，並利用mean DCF fluorescence的值評估活性氧產生的程度。此外，並結合細胞質分裂阻斷之微核分析 (CBMN)輔以DAPI染色的技術下，計算凋亡細胞、微核細胞、核質間橋等細胞的數量，進行基因毒性的檢測。
實驗結果: 五種不同的牙本質黏著劑對中國倉鼠卵巢細胞產生了不同程度的細胞毒性影響。計算TC50所獲得的細胞毒性排名依序是：(D)>>(E)≧(C)>(A)>(B)。在高濃度的條件下，五種牙本質黏著劑皆會誘導sub-G0/G1值的增加。利用Modfit軟體進行細胞週期的評估，則可以看到在24小時的給藥時間下，B.C.D組出現顯著的G2/M期細胞週期停滯；A.C.D組則出現G0/G1期停滯的現象。而利用mean DCF fluorescence偵測到的活性氧含量，A,C,D,E在濃度為1/40 (%)時與其他濃度出現顯著差異；B組則在1/40 (%)和1/20(%)較其他濃度出現統計上的顯著差異。基因毒性的測試結果，誘導凋亡細胞產生的比例與細胞存活率實驗獲得一致性的結果；較高的微核頻率則發生在含有較小奈米填料的組別 (B,C,D)；核質間橋的數量則伴隨微核細胞頻率的增加，也顯示增加的趨勢。核質間橋的出現一方面代表了染色體的修復，另一方面也顯示了基因受損的程度。
結論: 五種不同的牙本質黏著劑分別產生了不同程度的細胞毒性影響，這可能與牙本質黏著劑內含的各種成分有關。細胞毒性可能來自於成分中活性氧的產生，而誘導細胞凋亡和細胞週期的停滯所致。由我們的實驗結果得知，奈米填料的存在與否將不是決定毒性大小的唯一因素，但是奈米填料的出現，卻有可能提高染色體的損傷或遺失，導致細胞中微核及核質間橋的增加，而此結果我們推論奈米填料的出現可能引致進一步的基因毒性。這些結果與進一步的發現將對於臨床復型牙科在牙本質黏著劑的運用與瞭解上有實質性的幫助。

關鍵字

細胞凋亡 ； 細胞週期 ； 中國倉鼠卵巢細胞 ； 細胞毒性 ； 牙本質黏著劑 ； 奈米 ； 活性氧

並列摘要

Objectives: To compare the cytotoxicity & genotoxicity of five dentin bonding agents (DBAs), including three nano-DBAs (DBA-B:Voco Futurabond NR, DBA-C: 3M Singlebond 2, and DBA-D:Dentsply Prime & Bond NT) and two non-nano-DBAs (DBA-A:Voco Solobond M and DBA-E:Dentsply Xeno III) by using Chinese hamster ovary (CHO-K1) cells.
Methods: CHO-K1 cells were exposed to different concentrations of DBAs (1/40000-1/20, v/v, %) and negative control(DMSO) for 24 hrs. Cytotoxicity of DBAs was evaluated by colony formation efficiency. For cell cycle analysis and reactive oxygen species (ROS) production, CHO-K1 cells were treated with DBAs and then stained with propidium iodide (40ug/ml) or dichlorofluorescein–diacetate (DCFH-DA), respectively, and finally subjected for flow cytometric analysis. The percentage of cells in G0/G1, S, G2/M and sub-G0/G1 phases were determined. The mean DCF fluorescence was used to evaluate cellular ROS levels. Additionally, genotoxicity of DBAs was evaluated by CBMN & DAPI staining.
Results: DBAs showed differential cytotoxicity to CHO-K1 cells. The potency of cytotoxicity was (D)>>(E)≧(C)>(A)>(B). All five DBAs induced sub-G0/G1 cell population at higher concentrations. After exposure of CHO-K1 cells to B.C.D for 24 h, marked G2/M cell cycle arrest was noted, whereas A.C.D induced G0/G1 cell cycle arrest. DBAs also induced ROS production as indicated by an increase in mean DCF fluorescence after exposure to DBAs (A,C,D,E at a concentration of 1/40 (%), and B at a concentration of 1/40(%) or higher,1/20(%)). About genotoxicity, significant increase of MN frequency & NPB was noted in DBAs(B,C,D), including composition of nano-filler.
Conclusions: These five DBAs showed differential cytotoxicity to CHO-K1 cells. Cytotoxicity can be due to induction of apoptosis and arresting of cell cycles by components in DBAs and associated with ROS production. Absence or presence of nano-fillers is not the major cytotoxic factor for these DBAs, but significant increase of MNs & NPB could represent DNA loss or damage from nano-filler of DBAs. These results are useful for clinical operative restorative procedures.

並列關鍵字

Apoptosis ； Cell cycle ； CHO-K1 cells ； Cytotoxicity ； Dentin bonding agent ； Nano-materials ； Reactive oxygen species

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