褪黑激素對於人類牙周韌帶細胞成骨分化的影響

Abstract

褪黑激素是由人體腦內松果體所分泌的荷爾蒙，與晝夜節律及睡醒周期調節 有關，褪黑激素也具有免疫調節及抗氧化特性，近年研究更顯示褪黑激素能有效 提升間質幹細胞的活性並促進其成骨分化。本研究目的是探討褪黑激素是否影響 牙周韌帶細胞生長與成骨分化，並使細胞暴露於適量過氧化氫後，評估褪黑激素 能否幫助細胞抵抗氧化壓力及其可能的作用路徑。從矯正病人拔除的小臼齒培養 初代牙周韌帶細胞，在不同褪黑激素濃度作用後，進行細胞生長與成骨分化測試， 並利用反轉錄聚合酶連鎖反應分析可能作用基因及褪黑激素對其 mRNA 表現的調控。 另外將細胞暴露於亞致死濃度的過氧化氫，再給予褪黑激素作用後，檢測細胞活 性及成骨分化表現。除上述初代培養細胞之外，本研究另以永生化人類牙周韌帶 細胞株同步進行實驗。本研究發現濃度介於 0.01 uM~10 uM 褪黑激素對於初代牙 周韌帶細胞的生長活性，並無明顯影響，但在代數較大的初代人類牙周韌帶細胞， 加入 100 uM 褪黑激素會使細胞活性顯著下降，顯示對細胞生長有抑制作用。相較 於初代培養細胞，永生化人類牙周韌帶細胞對褪黑激素的反應比較不明顯。在成 骨分化表現，包括鹼性磷酸酶(ALP)活性檢測、細胞外基質礦化染色及定量分析， 都顯示褪黑激素在適當濃度(1 uM 與 10 uM)下，具有促進初代牙周韌帶細胞成 骨分化的效果。在沒有成骨誘導因子存在下，永生化人類牙周韌帶細胞即具有明 顯成骨分化能力，在 1 uM 或 10 uM 褪黑激素作用後，鹼性磷酸酶(ALP)活性微幅 上升，但是細胞外基質礦化染色的表現，卻顯示褪黑激素會造成抑制作用。接受 亞致死濃度的過氧化氫作用二小時並經長期培養後，牙周韌帶細胞的成骨分化表 現顯著下降。如果在過氧化氫作用後，給予適當濃度褪黑激素作用，可有效幫助 細胞恢復成骨分化能力，另外，在過氧化氫作用後給予褪黑激素的組別，其細胞 形態、生長密度、衰老比例，對照加入過氧化氫但沒有褪黑激素的組別，都會更 接近沒有加入過氧化氫的控制組，可見褪黑激素具有幫助細胞免於氧化壓力傷害 的作用。至於成骨分化相關基因的 mRNA 表現，相較於常規培養條件，接受成骨 分化誘導的初代人類牙周韌帶細胞的 ALP 基因 mRNA 表現明顯上升，而且在褪黑 激素存在下，上升幅度更為顯著，推測褪黑激素促進初代人類牙周韌帶細胞成骨 分化與 ALP 基因被向上調控有關。但永生化人類牙周韌帶細胞在成骨分化相關基因表現並沒有明顯變化趨勢。本研究證實褪黑激素具有促進人類牙周韌帶細胞成 骨分化的作用，並且能幫助細胞抵抗過氧化氫造成的氧化壓力。

Melatonin, a hormone secreted by pineal gland, exhibits modulating actions on many physiological functions including sleep-wake cycle, circadian rhythms, and anti-oxidation effect. Recent researches reveal that melatonin can enhance the bioactivity and osteogenic differentiation of mesenchymal stem cells. The study aimed to investigate whether melatonin enhance osteogenic differentiation of human periodontal ligament (PDL) cells and if melatonin can protect the cells from hydrogen peroxide-induced oxidative stress. The primary human periodontal ligament cells were isolated and cultured from healthy premolars extracted from young adult orthodontic patients. The immortalized human periodontal ligament cells purchased from abm company were parallel examined in this study. The alamar blue assay, measurement of alkaline phosphate activity, and alizarin red S staining were performed after treatment with melatonin. The mRNA expression levels of osteogenic genes were analyzed using RT-PCR technique. Moreover, the primary periodontal ligament cells were treated with melatonin after exposure to 200 uM hydrogen peroxide for 2 hr. The effect of melatonin in protecting cells from oxidative stress was evaluated by using a senescence associated beta-galactosidase assay. Our results showed that the melatonin (0.01 ~ 100 uM) did not affect the proliferation of primary human PDL cells, whereas 100 uM melatonin significantly inhibited the cell proliferation. Moreover, the melatonin (1 and 10 uM) significantly enhanced the alkaline phosphatase activity and mineralization of extracellular matrix the in primary PDL cells. Immortalized PDL cells could express extracellular matrix mineralization even in the absence of osteogenic factors. However, the extracellular matrix mineralization was inhibited by the melatonin (1 and 10 uM) in the immortalized PDL cells. After exposure to hydrogen peroxide, the osteogenic differentiation in primary PDL significantly decreased, and the adverse effect could be partly abolished by the treatment with melatonin. In addition, melatonin could upregulate the mRNA expression of alkaline phosphatase in primary human PDL cells cultured with osteogenic medium. However, the mRNA expressions of the osteogenic related genes did not show a consistent trend of change in immortalized PDL cells. In conclusion, melatonin can enhance the osteogenic differentiation of primary human PDLs and increase the cellular resistance against oxidative stress.