檳榔素與尼古丁對造牙骨質細胞影響之研究

The Study of the Effects of Nicotine and Arecoline in Murine Cementoblast

陳怡睿(Yi-Juai Chen)

指導教授 ： 蔡吉政

中山醫學大學/口腔醫學院/牙醫學系博士班/博士(2015年)

https://doi.org/10.6834/CSMU.2015.00148

摘要

臨床研究與流行病學的證據都顯示吸菸與嚼食檳榔對牙周組織有害。嚼食檳榔是台灣特有的文化，而多數嚼食檳榔者同時有吸菸的習慣。因此，在探討嚼食檳榔對牙周病之影響時，須同時考慮吸菸所造成的影響。檳榔子(areca nut)中主要的生物鹼(alkaloids)是檳榔素(arecoline)。它抑制了牙周細胞的生長、附連及增殖。尼古丁(nicotine)是吸菸產物中主要的生物鹼。它則抑制了牙周細胞的生長、增殖及麩胱甘肽(glutathione)與蛋白質的合成。然而，檳榔素和尼古丁對牙周硬組織，特別是牙骨質的影響所做的研究並不多。實驗利用小鼠永生造牙骨質細胞株OCCM.30 (murine immortalized cementoblast cell line)，探討檳榔素與尼古丁對造牙骨質細胞的抑制作用與致細胞病變效應(cytopathologic effects)。在檳榔素的部分，利用MTT測定法(tetrazolium bromide reduction assay)分析細胞毒性；利用transwell測定法評估細胞遷移；以von Kossa染色檢測體外試驗中礦化小結(mineral nodule)的形成；以受質分析法(substrate assay)檢測檳榔素對鹼性磷酸酶(alkaline phosphatase，簡稱ALP)活性的影響來評估細胞分化；使用ELISA (enzyme linked immunosorbent assay)評估骨保護素(osteoprotegerin，簡稱OPG)的產物。在尼古丁的部分，利用MTT法測定細胞存活率；以受質分析法檢測尼古丁對ALP活性的影響來評估細胞分化；使用ELISA和Griess試劑分別評估前列腺素E2 (prostaglandin E2，簡稱PGE2)和一氧化氮(nitric oxide，簡稱NO)產物；以西方墨點法(western blot)評估誘導型一氧化氮合成(inducible nitric oxide synthase，簡稱iNOS)之結果。實驗結果顯示，檳榔素與尼古丁對造牙骨質細胞的細胞毒性均呈現劑量依賴性(dose-dependent manner) (p<0.05)，另外，檳榔素表達的細胞毒性也呈現時間依賴性(time-dependent manner) (p<0.05)；檳榔素隨劑量的增加造成細胞遷移的衰減(p<0.05)；體外試驗第八天，經檳榔素處理過的造牙骨質細胞之生物礦化作用明顯被抑制了；檳榔素與尼古丁皆會隨時間的增加抑制ALP的活性(p<0.05)；檳榔素還會隨劑量的增加而減少OPG的分泌(p<0.05)；此外，尼古丁對PGE2分泌的增加與誘導NO生成及iNOS的表達皆呈現劑量依賴性(p<0.05)。以上的結果，證實檳榔素能夠抑制造牙骨質細胞的細胞成長、遷移和分化，而嚼食檳榔可能更容易造成牙周組織的破壞且降低牙周治療的過程中組織再生的反應。此外，實驗也證實尼古丁能抑制造牙骨質細胞的成長和分化，也能夠藉由增加PGE2的分泌以及iNOS的表達來誘發發炎反應，造成牙周硬組織更大的損傷。

關鍵字

檳榔素 ； 尼古丁 ； 造牙骨質母細胞 ； 細胞毒性 ； 細胞遷移 ； 細胞分化 ； 鹼性磷酸酶前列腺素E2 ； 一氧化氮

並列摘要

Studies have supported a higher prevalence of periodontal disease among areca quid chewers than non-chewers. However, few studies have stated the effects of arecoline, the major alkaloid of areca nut, on periodontal tissues. Cigarette smoking is an important risk factor in the pathogenesis of periodontal disease. Little is known about the effect of nicotine, the major component of cigarette smoke, on cementoblasts. Aims of these studies were to investigate the inhibitory effects of arecoline and the cytopathologic effects of nicotine on murine immortalized cementoblast cell line (OCCM.30). To investigate the inhibitory effects of arecoline, cytotoxicity was judged using tetrazolium bromide reduction (MTT) assay. Cell migration was evaluated by transwell assay. In vitro, mineral nodule formation was assayed by von Kossa staining. Cell differentiation was examined by alkaline phosphatase (ALP) assay by substrate assay. The production of osteoprotegerin (OPG) was evaluated using enzyme linked immunosorbent assay (ELISA). And to investigate the cytopathologic effects of nicotine, cell viability was judged by using MTT assay. Cell differentiation was examined by ALP assay. The production of prostaglandin E2 (PGE2) and nitric oxide (NO) were evaluated using ELISA and Griess reaction, respectively. Inducible nitric oxide synthase (iNOS) was evaluated by western blot. Arecoline demonstrated cytotoxicity to cementoblasts in a dose-dependent and time-dependent manner (p<0.05). Arecoline attenuated cell migration in a dose-dependent manner (p<0.05). Arecoline treatment markedly suppressed cementoblast-mediated biomineralization in vitro compared to untreated cells at day 8. Arecoline was found to inhibit ALP activity in a time-dependent manner (p<0.05). In addition, arecoline decreased the secretion of OPG in a dose-dependent manner (p<0.05). Nicotine demonstrated cytotoxicity to cementoblasts in a dose-dependent manner (p<0.05). Nicotine was found to inhibit ALP activity in a time-dependent manner (p<0.05). In addition, nicotine increased the secretion of PGE2 in a dose-dependent manner (p<0.05). Nicotine was found to induce NO generation and iNOS expression in a dose-dependent manner (p<0.05). Taken together, these results suggest that arecoline could inhibit cell growth, migration, and differentiation in cementoblasts. Areca quid chewers might be more susceptible to the destruction of periodontium and less responsive to regenerative procedure during periodontal therapy. In addition, nicotine could inhibit cementoblast growth and differentiation. It could also induce the inflammatory effects by the augmentation of PGE2 secretion and iNOS/NO expression.

並列關鍵字

arecoline ； cementoblasts ； cytotoxicity ； migration ； differentiation ； nicotine ； alkaline phosphatase ； prostaglandin E2 ； nitric oxide

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