由流行病學研究報告指出吸煙會促進牙周病的進行及延遲牙周治療的癒合反應，但從文獻回顧中發現鮮少有研究探討香煙對牙周組織的破壞機轉，所以本研究以組織培養法，培養人類牙周韌帶造纖維母細胞，測定尼古丁對細胞生長、細胞蛋白質合成、GSH之含量、加入一些自由基捕捉劑或抗氧化劑，以預防醫學的角度，試圖找到一些解毒的藥物，達到化學預防(chemoprevention)的目的；另外以逆反轉聚合媒鏈反應探討尼古丁對牙周韌帶造纖維母細胞所產生cellular stress，是否會經由c-fos之pathway，從分子生物學的角色找到early gene調控的機轉，探討香煙的主要成份尼古丁對牙周韌帶造纖維母細胞的抑制機轉。牙周韌帶造纖帶母細胞係來自於矯正拔牙的牙齒。實驗結果顯示：尼古丁會對人類牙周韌帶造纖帶母細胞產生細胞毒性作用，此一現象會隨著濃度、作用的時間增加而增加 (p<0.05)；且尼古丁也會明顯地抑制細胞增殖及降低蛋白質的合成，尼古丁的濃度在50 mM及200 mM時分別會抑制48 % 及86 % 人類牙周韌帶造纖維母細胞的生長，在10 mM濃度的尼古丁會明顯地抑制蛋白質合成量約為控制組的44 % (p<0.05)；加入細胞外2-oxothiazolidine-4-carboxylic acid (OTZ)可以保護細胞免於尼古丁誘發的細胞毒性，但在加入50 mM BSO (buthionine sulfoximine) 後其可加強尼古丁造成的細胞毒性；superoxide dismutase及catalase並不會影響由尼古丁引起的細胞毒性反應；尼古丁在5 mM時會明顯地消耗細胞內glutathione (GSH) (p＜0.05)，同時glutathione disulfide也一起跟著減少，由這結果顯示是硫醇(thiol)的消耗，而非因氧自由基引起尼古丁的細胞毒性機制。此外本實驗亦首次發現尼古丁會誘發人類牙周韌帶造纖維母細胞c-fos早期反應基因的表達，但事先加入OTZ處理後會降低c-fos基因的表達，並且細胞經由BSO事先處理後會加強c-fos 基因的表達，這些結果顯示尼古丁在人類牙周韌帶造纖維母細胞中是經由活化c-fos早期反應基因路徑，並且細胞內硫醇濃度的高低可調節由尼古丁誘導c-fos基因的表達。

The use of tobacco products significantly contributes to the progression of periodontal disease and poor response to healing following periodontal therapy. The purpose of this study was to determine the pathobiological effects of nicotine, a major component of cigarette smoking, on human periodontal ligament fibroblasts (PDLFs) to elucidate its role in periodontal destruction associated with its use. Human PDLFs were derived from healthy individuals undergoing extraction for orthodontic reasons. At a concentration higher than 2.5mM, nicotine was found to exhibit cytotoxic to human PDLFs (p<0.05). Nicotine also significantly inhibited cell proliferation and decreased protein synthesis in a dose-dependent manner. At concentrations of 50 and 200 mM, nicotine suppressed the growth of PDLFs with 48% and 86% (p<0.05), respectively. A 10 mM concentration level of nicotine significantly inhibited the protein synthesis to only 44 % of these in the untreated control (p<0.05). Furthermore, the effects of anti-oxidants were added to search for the possible mechanism of action, as well as a method for the prevention, of cigarette smoking-associated periodontal diseases. The addition of OTZ, a precursor of cysteine that metabolically promotes GSH synthesis, acted as a protective effect on the nicotine-induced cytotoxicity. However, superoxide dismutase and catalase did not decrease the nicotine-induced cytotoxicity. In contrast, the addition of BSO, a cellular GSH synthesis inhibitor, enhanced the nicotine-induced cytotoxicity. In addition, nicotine significantly depleted intracellular GSH in a dose-dependent manner (p<0.05). At a concentration of 5 mM and 20 mM, nicotine depleted about 22.2 % and 56 % of GSH, respectively. The exposure of quiescent human PDLFs to nicotine resulted in the induction of c-fos mRNA expression. The peak of c-fos mRNA levels induced by nicotine was 5 mM at 2 h incubation period. Kinetic investigations of c-fos mRNA expression in nicotine-treated cells revealed a rapid accumulation of the transcript, a significant signal first detectable after 30 min of exposure. This increase was transient and the level of c-fos mRNAs returned rapidly to that of control cells by 8 h. OTZ pretreatment drcreased in c-fos mRNA level and BSO pretreatment enhanced in c-fos mRNA level after exposure to nicotine. The levels of nicotine tested inhibited cell growth, proliferation, and protein synthesis on human PDLFs. This suggests that nicotine itself might augment the destruction of periodontium associated with cigarette smoking. In addition, these inhibitory effects were associated with intracellular thiol levels. Factors that induce glutathione synthesis of human PDLFs may be used for further chemoprevention of cigarette smoking-related periodontal diseases. In addition, c-fos gene expression might be one signal transduction pathway linked to the induction of early response genes by cigarette smoking. These results suggest that the nicotine-dependent stress-specific expression of the c-fos gene correlates with cellular thiol levels in human PDLFs.

中文摘要-----------------------------------------1
英文摘要-----------------------------------------3
前言---------------------------------------------6
材料與方法---------------------------------------19
實驗結果-----------------------------------------23
討論---------------------------------------------26
圖表與圖表說明-----------------------------------33
參考文獻-----------------------------------------49

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