探討 BisGMA 刺激小鼠巨噬細胞產生 PGE2 與細胞毒性–經由COX-2、cPLA2、MAPK family 路徑之研究

Bisphenol A-glycidyl-methacrylate stimulates prostaglandin E2 production and cytotoxicity in macrophages via cytosolic phospholipase A2, cyclooxygenase 2 and mitogen-activated protein kinases family

陳楓宜(Feng-Yi Chen)

指導教授 ： 關宇翔 ； 李怡靜

中山醫學大學/醫學院/醫學研究所/碩士(2013年)

https://doi.org/10.6834/CSMU.2013.00051

摘要

複合樹酯單體為應用廣泛之生醫材料，普遍被用於牙科補牙與骨質黏著，因具有黏附性優異、聚合迅速且美觀之特點，近幾年來大量使用於牙科補牙材料，但有文獻指出複合樹脂單體聚合後可能再度釋出而導致發炎反應之產生。
Bisphenole-glycidyl methacrylate (BisGMA) 為其中之一項材料，在最近之文獻中也有指出BisGMA 會誘導人類牙髓細胞與巨噬細胞之細胞毒性、遺傳毒性與發炎反應，且會誘導巨噬細胞之促發炎因子產生，如 tumor necrosis factor-α (TNF-α)、 interleukin-1β (IL-1β) 和 interleukin-6 (IL-6) 等。Prostaglandin E2 (PGE2) 在免疫反應中為調節發炎的一個關鍵，但是由 BisGMA 誘導之巨噬細胞活化中 PGE2 的機制還不是很明確。
因此本研究的目的在於評估由BisGMA對於小鼠之巨噬細胞所誘導之 PGE2 訊息傳遞路徑。利用酵素免疫分析法 (enzyme-linked immunosorbent assay；ELISA) 確認 PGE2 之表現會隨著 BisGMA 之濃度提高而上升，再利用西方墨點法 (western blot) 觀察 PGE2 之上游蛋白磷酸化的表現，包括 cytosolic phospholipase A2 (cPLA2) 、cyclooxygenase-2 (COX-2) 以及 mitogen-activated protein kinase (MAPK) pathway中之 extracellular-signal-regulated kinase 1/2 (ERK1/2)、c-Jun N-terminal kinase (JNK) 和 p38 mitogen-activated protein kinase (p38) pathway。
結果顯示，BisGMA會活化 ERK1/2 pathway，包括 mitogen-activated protein kinase kinase 1/2 (MEK1/2)、ERK1/2 和 ETS domain-containing protein (Elk)，且有劑量效應關係。BisGMA 也會活化 p38 pathway，包括mitogen-activated protein kinase kinase 3/6 (MEK3/6)，p38 和 MAPK-activated protein kinase 2 (MAPKAPK2)； JNK pathway，包括 mitogen-activated protein kinase kinase 4 (MEK4) ， JNK 及 c-Jun ，也是一樣之情況。隨後再加入 ERK1/2、p38 和 JNK 磷酸化之抑制劑 - U0126、SB203580、SP600125、AACOCF3，再度確認 BisGMA 會誘導 ERK1/2、p38、JNK 和 cPLA2 磷酸化之產生。以上結果顯示 BisGMA 誘導的巨噬細胞活化是通過 COX-2 的表達， cPLA2 的磷酸化和 ERK1/2、p38、JNK pathway 來調控 PGE2 之表現。此外，發現BisGMA 也會經由MAPK family 造成 cysteine-aspartic proteases (caspase) 3、8、9 的活化導致DNA 損傷，進而導致apoptosis的產生。

關鍵字

複合樹酯單體 ； 巨噬細胞 ； DNA 損傷 ； 細胞毒性 ； 遺傳毒性 ； 發炎反應 ； MAPK 家族

並列摘要

One of the materials, bisphenole-glycidyl methacrylate (BisGMA), recently has been point out in some studies which can cause cytoxicity, genotoxicity and inflammation in human dental pulp cells and macrophages. For instance, BisGMA induced the generating of proinflammatory cytokines such as tumor necrosis factor-α(TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). In particular, prostaglandin E2 (PGE2) plays an important role in immunomodulatory; however, the mechanism of BisGMA induces PGE2 activation in macrophage has not been clearly defined.
Therefore, the purpose of this study is to evaluate the inductive effects of BisGMA induce PGE2 activation signal pathway in mice macrophage (RAW264.7). Throughout this experiment,enzyme-linked immunosorbent assay (ELISA) was used to confirm that BisGMA does increase PGE2 level in concentration-dependent manner. Furthermore, western blots were used to analysis the PGE2 protein phosphorylation levels: including cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2) and also ERK1/2, JNK and p38 pathways in the MAPK pathway. The result shown that, BisGMA will activate, (1) extracellular-signal-regulated kinase 1/2 (ERK1/2) pathways including mitogen-activated protein kinase kinase 1/2 (MEK1/2), ERK1/2 and ETS domain-containing protein (Elk), (2) p38 pathway, including mitogen-activated protein kinase kinase 3/6 (MEK3/6), and MAPk-activated protein kinase 2 (MAPKAPK2), (3) JNk pathway, including mitogen-activated protein kinase kinase 4 (MEK4), c-Jun N-terminal kinase (JNK) and c-Jun, in dose-dependent manner. Pretreatment U0126 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor) and AACOCF3 (cPLA2 inhibitor), reconfirmed that BisGMA will induce the phosphorylation of ERK1/2、p38、JNK and cPLA2.
As shown above, the generating of PGE2 was induced by BisGMA, which involved the phosphorylation of cPLA2 via ERK1/2, p38 and JNK pathways in RAW264.7 macrophages. Moreover, BisGMA caused DNA damage which activated by cysteine-aspartic proteases (caspase) 3、8、9 then led to apoptosis.

並列關鍵字

BisGMA ； PGE2 ； cPLA2 ； COX-2 ； MAPK family ； caspase-3 ； caspase-8 ； caspase-9 ； apoptosis ； DNA damage

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