香菸煙霧萃取物對於人類牙齦纖維母細胞之發炎反應

Effects of cigarette smoke extract on inflammation response of human gingival fibroblasts

邱鈺翔(Yu-Hsiang Chiou)

指導教授 ： 王彥雄

高雄醫學大學/口腔醫學院/牙醫學系碩士班/碩士(2019年)

摘要

研究目的: 慢性牙周病是目前口腔疾病中最常見的慢性疾病之一，而抽菸被認為是導致牙周疾病的重要影響因子。以往的研究發現香菸中的尼古丁會引起細胞發炎因子表現量上升，但中間傳遞的機轉仍然不明。而香煙煙霧裡面含有7,000多種化學成分，所以與純尼古丁相比，其他成分對於口腔的影響可能不一樣。本篇實驗將以香菸萃取物對於人類牙齦纖維母細胞之發炎機轉做詳細探討。
材料及方法: 使用人類牙齦纖維母細胞，加入5%~20%之香菸萃取物後，處理一至三天，進行細胞存活率分析 (MTT assay)。再以不同濃度 (2.5%和5%) 之香菸萃取物，處理細胞一天，針對白細胞介素6、8及TLR-4，以及加入P38、ERK和AKT抑制劑後萃取細胞RNA並進行即時聚合酶連鎖反應 (qPCR) 觀察其表現量。蛋白質表現量則取濃度2.5%之香菸萃取物加入細胞培養，使用西方墨點法 (Western blot test) 觀察蛋白表現。
結果:細胞存活率分析發現，隨著香菸萃取物濃度上升而細胞存活率下降。mRNA表現發現白細胞介素6、8及TLR-4在加入香菸萃取物後表現量會上升。使用西方墨點法發現磷酸化的P38、AKT及ERK有增加的現象，且分別加入P38、ERK和AKT抑制劑皆發現白細胞介素6和8的表現量會被顯著抑制。
結論: 香菸萃取物對於牙齦纖維母細胞有可能是藉由TLR-4及P38、ERK、AKT路徑導致白細胞介素6和8之表現上升。

關鍵字

香菸煙霧萃取物 ； IL-6 ； IL-8

並列摘要

Objectives: Chronic periodontitis is one of the most common oral inflammatory diseases, and cigarette smoking is considered to be an important etiologic factor. Nicotine is a key active ingredients in cigarette. Previous studies demonstrated that nicotine has been shown to increase the expression of inflammatory cytokines in human gingival fibroblast, but the pathway is still unknown. In fact, cigarette contains more than 7,000 chemical components that the smoking-induced inflammatory response might be very different compared to the pure nicotine-induced response. In this study we investigated the mechanism of the cigarette smoke extracts (CSE)-induced inflammatory response in human gingival fibroblasts (HGFs).
Material and methods: HGFs were treated with 5%~20% of CSE for one to three days. Cell viability was analyzed by MTT assay. The mRNA level of interleukin-6 (IL-6), IL-8, and Toll like receptor-4 (TLR-4) was detected by qPCR. The protein expressions of P38, ERK, and AKT were monitored by Western blot.
Results: The survival rate of HGFs was decreased when the concentration of CSE increased. The mRNA levels of IL-6 and IL-8 were significantly increased after CSE treatment. The signaling pathways of P38, AKT and ERK were also activated by CSE treatment. All the inhibitors of P38, AKT and ERK inhibited the CSE-induced expression of inflammatory cytokines.
Conclusion: CSE may activate TLR-4 and trigger P38, ERK, and AKT signaling pathways to up-regulate the expression of inflammatory cytokines in HGFs.

並列關鍵字

Cigarette smoke extract ； IL-6 ； IL-8

參考文獻

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