丁酸對MG-63類骨瘤細胞之細胞週期、細胞型態以及活性氧產生的影響

Effect of Butyrate on cell cycle, cell morphology and Reactive Oxygen Species production of MG-63 osteoblasts

廖敏為(Min-Wei Liao)

指導教授 ： 鄭景暉

國立臺灣大學/牙醫專業學院/臨床牙醫學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.02833

摘要

實驗目的: 丁酸(丁酸)是一種常見的厭氧性葛蘭氏陰性菌(anaerobic Gram-
negative bacteria)的代謝產物，常見於口腔致病菌，亦常見於牙髓以及牙周致病菌中。本篇研究的目的是探討丁酸對MG-63類骨瘤細胞的毒性、訊息傳遞與細胞週期的進行的過程中所扮演的角色。藉此了解此牙髓與牙周致病菌常見的毒素產物對於牙齒周圍骨組織的影響，有助於釐清牙根尖齒槽骨與牙周環境發炎和修復的生理病理過程。
實驗方法: 本研究以丁酸在不同的刺激濃度以及刺激時間作用於MG-63類骨瘤細胞，觀察其細胞反應以及細胞週期相關因子的變化。
實驗結果:
1.丁酸 對於 MG-63 細胞的毒性方面，在本實驗設定之濃度(0- 16 mM)與刺激時間內對於 MG-63 類骨瘤細胞的細胞型態與生長來說並沒有明顯的抑制現象，在光學顯微鏡下以及電子顯微鏡下MG-63細胞的外型並沒有顯著的變化。
2.在細胞存活率分析的結果顯示在高濃度(尤其是16mM)的丁酸的刺激下，細胞的生長分化顯著受到抑制。
3.加入丁酸對於MG-63細胞的訊息傳遞與細胞週期方面，它能夠影響細胞週期在G2期進入mitosis的過程，相關的調控因子如Cdc2、Cdc25C以及Cyclin-B1的蛋白質反應; Cdc25C以及Cyclin-B1的mRNA表現都會受到丁酸的影響而下降，隨著丁酸濃度上升，G2期進入mitosis的停滯現象會變明顯。
4.MG-63細胞在較高濃度丁酸的刺激下，活性氧以及p21的產量會顯著的提升，亦可能會造成細胞週期的停滯。
結論: 厭氧性葛蘭氏陰性菌的重要產物丁酸會造成MG-63細胞的細胞週期停滯，尤其在進入分裂期前，隨濃度上升影響更鉅，顯示在牙周發炎以及牙根尖發炎中細菌所產生的丁酸可能會影響周圍骨細胞，進而延滯周圍骨頭的修復。亦即，丁酸的毒性與活性氧過度產生以及細胞週期相關之蛋白質與RNA表現有關。

關鍵字

丁酸 ； MG-63類骨瘤細胞 ； 細胞週期 ； 細胞生長 ； 活性氧 ； 根尖病變 ； 根管致病菌

並列摘要

Aim:
Butyric acid, one of major byproducts produced by anaerobic Gram-negative periodontal and root canal micro-organism and is easliy detected in gingival crevicular fluid from human periodontal pockets. The aim of this study is to evaluate the role of butyric acid in the cytotoxicity, signaling pathway and cell cycle of the MG-63 osteoblast cell-line. By understanding the role of butyric acid in MG-63 osteoblast cell-line, we can surmise the effect of this toxicant in human alveolar bone and the possible reaction while osteoblasts encounter butyric acid and the possible progression of bone healing.

Material and Methods:
MG-63 osteoblast cells were treated with butyric acid in different concentration and different stimulation time. The MG-63 cell morphology and cell cycle-related factors were evalutated: Cell morphology was observed under optical microscope and scanning eletron microscope. MTT assay was used to appraise the cell viability. Changes in mRNA expression was determined by reverse- transcriptase polymerase chain reaction (RT-PCR). The protein level was evaluated by western blotting.

Results:
MG-63 cell under the treatment of butyric acid (range: 1mM-16mM) showed no significant change in cell morphology. The cell viability was significantly suppressed in high concentration group(16 mM) of butyric acid. Expression in protein level of Cdc2, Cdc25C and Cyclin-B1 were decreased as the concentration of butyric acid increased. The expression of the mRNA level of Cdc25C and Cyclin-B1 were decreased as the concentration of butyric acid increased. The ROS and the p21 expression of MG-63 cell were elevated in the groups of high concentration of butyric acid.

Conclusions:
Many factors could affect the progress of cell cycle, one of the important by-products of Gram-negative bacteria- butyric acid may take part in cell cycle, high concentration of butyric acid might hinder the process of the cell cycle, especially in G2-mitosis phase. butyric acid might also elevate the production of ROS, which may affect the cell cycle of MG-63

並列關鍵字

butyric acid ； MG-63 osteoblasts ； cell cycle ； cell growth ； periapical lesion ； root canal microorganism

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