低能量雷射照射對人類牙周韌帶細胞periostin 表現的影響

Abstract

低階能量雷射照射可透過給予組織適當能量，促成抗發炎、止痛、促進傷口癒合等治療目的，近年來，低階能量雷射治療的臨床療效被應用於牙醫學的領域。細胞外間質蛋白periostin具有調控細胞附著、移動、分化等功能，其表現與牙周韌帶承受咬合力的能力及組織結構的穩定性關係密切。PLAP-1 (periodontal ligament - associated protein, PLAP-1)抑制牙周韌帶的細胞成骨分化潛力，可能與牙周韌帶能夠維持結締組織形態而免於鈣化的特性有關。 本研究探討低能量二極體雷射對人類牙周韌帶細胞活性及periostin與PLAP-1表現的影響。實驗細胞是取自齒顎矯正治療所拔除的小臼齒或智齒所培養的人類牙周韌帶細胞，雷射機型為波長970nm半導體雷射(Al-Ga-As，Arts K-Laser Professional 40E 970 nm)，照射參數設定為continuous wave mode，output power：500 mW， 照射時間為4秒( 5.20 J/cm2) 、8秒( 10.40 J/cm2)，照射探頭與culture plate底部距離為11 mm。雷射所引起的細胞效應分析包括: 細胞生長活性測試，periostin、PLAP-1基因的mRNA表現分析、以及periostin、Smad 2/3 蛋白質表現分析。 本研究結果顯示波長970nm之低階能量雷射照射會使牙周韌帶細胞的MTT活性明顯提升，隨著時間變化因能量密度而有不同改變。RT-PCR分析基因結果顯示在能量密度5.20 J/cm2照射一天後，periostin 及PLAP-1 mRNA表現增加。Western blot分析發現當細胞培養環境存在外加轉化生長因子TGF-β時，牙周韌帶細胞periostin表現會增強；單次低階能量雷射照射對牙周韌帶細胞之periostin的影響皆呈現先降低而後再回復至與control groups 相似的情形。重複雷射照射則在能量密度不同與照射天數不同相較於單次照射有增強或削弱的表現差異。本研究顯示低階能量雷射可能會強化牙周韌帶細胞的periostin 與PLAP-1表現進而影響牙周組織的平衡，但低階能量雷射照射是否促進TGF-β訊息路徑的活化並促成Smad 2/3磷酸化的作用尚待進一步探討。

關鍵字：低階能量雷射照射、人類牙周韌帶細胞、 Periostin、PLAP-1

Low level laser irradiation by giving proper dose application contributes to anti-inflammatory, analgesic, wound healing and other therapeutic purposes. In recent years, the clinical efficacy of the low level laser treatment is also applied to the field of dentistry. Periostin is an extracellular matrix protein highly expressed in periodontal ligament and is essential for periodontal tissue integrity. Its function is as follows regulation of cell adhesion, migration, differentiation and so on. Periostin expression is closely related with the ability of periodontal ligaments to withstand the bite force and the stability of PDL structure. PLAP-1/asporin (periodontal ligament- associated protein) plays specific roles in maintaining the homeostasis of the PDL as a negative regulator of cytodifferentiation and mineralization. This study investigated the effects of low level laser irradiation on the expression of periostin and PLAP-1 in human PDL cells. Experimental PDL cells were collected from human wisdom teeth or premolars extracted for orthodontic reasons. 970nm semiconductor laser models (Al-Ga-As, Arts K-Laser Professional 40E 970 nm) was used in the study , irradiation parameter was set to continuous wave mode, output power was500 mW, irradiation time was 4 seconds (5.20 J/cm2) , 8 seconds (10.40 J/cm2) , the distance between the tip of the laser handpiece and the button of the culture plate was 11mm. Analysis of low level laser irradiation on PDL cells included MTT assay, expression of periostin mRNA , PLAP-1 mRNA , and periostin, Smad2/3 protein. The results of this study showed that MTT activity of periodontal ligament cells under 970-nm low level laser irradiation increased significantly over time at different energy density. RT-PCR analysis of gene showed that after one-day low level laser irradiation at energy density of 5.20 J/cm2, periostin mRNA and PLAP-1 mRNA expression increased. Western blot analysis showed that periostin expression of periodontal ligament cells was enhanced with the presence of TGF-β. The impact of a single low-level laser irradiation on periostin protein expression in periodontal ligament cells were decreased first and then back to a similar situation compared with the control groups. The performance of repeated laser irradiation on periostin expression at different energy density and irradiation days were enhanced or weakened, which were different from those of single irradiation application. This study showed that low-level laser irradiation may enhance the expression of periostin and PLAP-1 in the periodontal ligament cells and thus may affect the homeostasis of periodontal tissue. However, whether the low level laser irradiation activate TGF-β signaling pathway and contribute to the phosphorylation of Smad2/3 remains to be further explored. Key word: low level laser irradiation、human periodontal ligament cell、Periostin、PLAP-1