- 學位論文
以低能量雷射照射高糖誘發人類牙齦纖維母細胞發炎與纖維化之效應
The effects of low-level laser irradiation on hyperglycemia-induced inflammation and fibrosis in human gingival fibroblasts
摘要
控制不良的高血糖症（hyperglycemia）通常伴有發炎反應，這可能是糖尿病患者更容易患牙周病的原因，高血糖症也會誘發纖維化反應，對人體組織造成損傷。在臨床應用上，低能量雷射透過光化學（photochemical）及光生物（photobiological）的效應，已被廣泛用作傷口癒合，抗發炎及疼痛解除的治療方法。我們先期的研究發現低能量雷射能抑制幹細胞受脂多醣（LPS）引發的發炎刺激反應，降低前發炎性細胞激素（pro-inflammatory cytokine）IL-1及IL-6基因的表現量。然而，低能量雷射對於患有牙周炎的糖尿病患者的治療效果仍然未知，對於抗纖維化的效果也需要進一步探討。 在本研究中，我們以高葡萄糖培養基（35 mM）模擬高血糖環境來培養人類牙齦纖維母細胞（Human gingival fibroblasts, HGFs），使用定量實時聚合酶鏈反應分析前發炎性細胞激素，包括腫瘤壞死因子-α（TNF-α），白細胞介素（IL）-1β，IL-6與IL-8和纖維化基因的α-平滑肌動蛋白（α-SMA），結締組織生長因子（CTGF）和乙型轉化生長因子（TGF-β）之基因表現量，來評估HGFs被引發的發炎與纖維化反應以及低能量雷射抑制發炎與纖維化反應的能力。本研究的結果顯示，用低能量雷射照射高葡萄糖培養基中HGFs，相對於在甘露醇培養基中培養的HGFs並沒有顯著差異。但是在高葡萄糖培養基中的HGFs，沒有低能量雷射照射的HGFs比用低能量雷射照射的HGFs有顯著更高的前發炎性細胞激素與纖維化基因的mRNA表現。為了評估低能量雷射作用的可能機制，也觀察到是通過調節cyclic AMP來降低高葡萄糖培養基中的HGFs前發炎性細胞激素的基因表現量，此外cyclic AMP調控機制對於纖維化基因也有影響。另一方面，我們也評估抗氧化劑（維生素C）對HGFs在高葡萄糖培養基中被氧化壓力引發的發炎反應的抑制效果，但在與低能量雷射的共處理中並沒有發現添加效應，這結果可能意味著低能量雷射以調節cyclic AMP信號通路，而不是活性氧（ROS）信號通路來降低高糖誘導的發炎反應。根據本研究的結果，LLLI可以在高糖環境中對HGFs具有抗炎作用，能對於糖尿病患者的牙周疾病的治療具有益處，也具有抑制細胞纖維化的效果。
並列摘要
Poorly controlled hyperglycemia often accompanied with inflammation. It may be the reason that the patients with diabetes are much more susceptible to periodontal disease. Hyperglycemia also induces the fibrotic response to damage tissues. Low-level laser irradiation (LLLI) has been used as a therapy for wound healing, anti-inflammation in many situations. In addition, recent researches suggested that LLLI inhibits the lipopolysaccharide (LPS)-stimulated inflammatory response. However, the therapeutic effect of LLLI on diabetes patients with periodontitis remains unknown and the effect to suppress fibrosis needs to study. In this study, we cultured the human gingival fibroblasts (HGFs) in high glucose medium (35 mM) in order to mimic a hyperglycemic environment, and then detected not only the induced inflammatory and fibrotic response but also the ability of LLLI on inhibition of inflammations and fibrosis by assessing the expression of pro-inflammatory genes, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and fibrotic genes, including α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and transforming growth factor β (TGF-β) with quantitative real-time polymerase chain reactions. The results of this study demonstrated no significant inflammatory response in HGFs cultured in mannitol medium and only treated by LLLI relative to in high glucose medium. Otherwise, HGFs cultured in high glucose medium without LLLI had significantly higher mRNA expression of pro-inflammatory cytokines and fibrotic genes than with LLLI. To determine the possible mechanism of LLLI action, we first observed that LLLI reduced of the expression level of pro-inflammatory cytokines in high glucose medium cultured HGFs through modulating cAMP signal and also to fibrotic genes. On the other hand, we also investigated the antioxidant (vitamin C) reduced the inflammatory effect from oxidative stress in high glucose medium cultured HGFs, but no additive effect was found in co-treatment with LLLI. This result might imply that LLLI may activate cAMP signaling pathway, but not reactive oxygen species (ROS) signaling pathway to reduce high glucose-induced inflammatory reaction. According to results of this study, LLLI may have an anti-inflammatory effect on HGFs in high glucose environment and a benefit for treatments to periodontal diseases in diabetes patients. It also suppresses the fibrotic response caused from high glucose condition.