- 學位論文
尼古丁對造牙骨質母細胞抑制機轉之研究
The inhibitory mechanisms of nicotine on cementoblasts
摘要
吸煙是導致牙周疾病的一個重要的危險因子，然而從文獻回顧中發現鮮少有研究探討香煙的主成分--尼古丁對造牙骨質母細胞的作用。所以本研究目的是探討尼古丁對造牙骨質母細胞的影響，以組織培養法，培養老鼠的造牙骨質母細胞株(簡稱：OCCM.30)，加入不同濃度的尼古丁作用於OCCM.30細胞株。利用alamar blue 還原試驗測定細胞存活率；用內管(Transwell)遷移和傷口癒合爬行試驗評估細胞增殖能力；另以酵素連結免疫吸附法(ELISA)測量細胞激素-第六型白細胞介素和腫瘤壞死因子-α蛋白質表現的濃度;並以半定量雙氯氫化螢光素雙乙酸鹽(DCFH-DA)螢光技術偵測細胞內產生的活性氧自由基(ROS)濃度。實驗結果顯示：尼古丁在濃度高於1.5 mM時，會對OCCM.30細胞產生細胞毒性作用(p＜0.05)；尼古丁也會明顯地抑制OCCM.30細胞爬行能力，此一現象會隨著濃度增加而增強(p＜0.05); 此外尼古丁會促進產生第六型白細胞介素和腫瘤壞死因子-α，此一現象亦會隨著濃度增加而增加(p＜0.05); 1 mM濃度的尼古丁會促進細胞內活性氧自由基的產生，此一現象會隨著時間增加而增加(p＜0.05)。由實驗結果顯示尼古丁會對OCCM.30細胞產生細胞毒性作用，其機轉係經由抑制OCCM.30細胞生長、爬行，及增加誘發發炎細胞激素分泌和活性氧自由基的產生。由現有研究結果推測尼古丁可使造牙骨質母細胞功能受損並導致不良作用，因而造成吸菸者的牙周組織較易受到破壞且對牙周治療的再生步驟較不易有良好的反應。
並列摘要
Cigarette smoking is an important risk factor in the pathogenesis of periodontal disease. However, little is known about the effect of nicotine, the major component of cigarette smoke, on cementoblasts. The aim of this study was to investigate pathological effects of nicotine on murine immortalized cementoblast cell line (OCCM.30). Cell viability was judged by using alamar blue reduction assay. Cell migration was evaluated by transwell and wound-healing assays. The protein concentrations of IL-6 and TNF-α were measured by using enzyme linked immunosorbent assay (ELISA). The semiquantitative DCFH-DA fluorescence technique was used to detect the intracellular level of reactive oxygen species (ROS). The concentrations of nicotine higher than 1.5 mM demonstrated cytotoxicity to cementoblasts (p < 0.05). Nicotine attenuated cell migration in a dose-dependent manner (p < 0.05). In addition, nicotine augmented the production of IL-6 and TNF-α in a dose-dependent manner (p < 0.05). The concentration of 1 mM nicotine enhanced the generation of intracellular ROS in a time-dependent manner (p < 0.05). Taken together, these results suggest that nicotine could inhibit cell growth and migration on cementoblasts. In addition, nicotine could also induce inflammatory cytokines and ROS generation. Within the limits of the present study, these results show that nicotine could impair cementoblast functions and cause adverse effects. Cigarette smokers might be more susceptible to the destruction of periodontium and less responsive to regenerative procedure during periodontal therapy.