- 學位論文
低能量雷射抑制檳榔鹼誘導口腔黏膜下纖維化之形成:離體試驗
Low-power laser irradiation inhibits arecoline-induced oral submucous fibrosis: an in vitro study
摘要
口腔黏膜下纖維化(Oral submucous fibrosis, OSF)是一種癌前病變的症狀，主要的特徵在於口腔黏膜下組織和深層結締組織的發炎反應以及纖維化現象。檳榔中的檳榔鹼---Arecoline已經被證實為口腔黏膜下纖維化的主要發病因子。 低能量雷射(Low-power laser irradiation, LPLI)已經被應用於幫助傷口癒合、治療發炎、幫助肌肉組織修復以及預防纖維化現象產生等方面，但其分子機制仍不清楚。此外，也沒研究探討低能量雷射是否能運用在檳榔鹼引發的口腔黏膜下纖維化的治療上。之前的研究顯示在人類脂肪幹細胞(human adipose-derived stem cells)中，低能量雷射可以透過cAMP訊號的路徑去抑制發炎反應。因此，在本論文中，我們研究有關於口腔黏膜下纖維化的致病機轉，以及評估低能量雷射對於檳榔鹼所引起的口腔黏膜下纖維化的治療效果。 在本實驗中，我們所使用的細胞為人類的牙齦纖維母細胞 (human gingival fibroblasts)。將細胞以檳榔鹼刺激，觀察在有無低能量雷射照射的情況下，纖維化基因 (fibrotic marker genes)，如α-SMA和CTGF的表現。以即時定量聚合酵素連鎖反應法(qRT-PCR)和西方點墨法 (Western blots)檢測基因的表現量，並以Reporter assay來測量CTGF基因的轉錄表現。 實驗結果顯示，檳榔鹼的刺激能促進人類牙齦纖維母細胞其 α-SMA和CTGF的mRNA和蛋白質表現量。而有趣的是，在本實驗結果中，我們發現在人類牙齦纖維母細胞中，無論是低能量雷射或是forskolin (adenylyl cyclase activator)，皆能抑制由檳榔鹼誘導的纖維化基因表現量，並且抑制CTGF的轉錄現象。此外，在檳榔鹼刺激人類牙齦纖維母細胞前，先以SQ22536 (adenylyl cyclase inhibitor) 對細胞做前處理，之後同樣給予低能量雷射的照射，會發現加入SQ22536有抑制低能量雷射的治療效益。 在本篇實驗結果顯示，低能量雷射有抑制纖維化的效果，且此作用可能是經由cAMP訊號路徑所產生。
並列摘要
Oral submucous fibrosis (OSF) is a potentially malignant disorder, which is characterized as a juxtraepithelial inflammatory reaction and progressive fibrosis in oral submucosa and deeper connective tissues. It has been reported that an alkaloid compound of areca nut, arecoline, is a major etiological factor in the development of OSF. Low-power laser irradiation (LPLI) has been applied for treating wound healing, inflammation, muscle tissue repair and preventing fibrosis. However, the therapeutic mechanism of LPLI and the potential therapeutic application for arecoline-induced OSF are still unclear. Previous studies indicated that LPLI may suppress inflammatory response in human adipose-derived stem cells via cAMP signaling pathway. Therefore, we investigate the molecular pathologic mechanisms of arecoline-induced OSF and evaluate the effects of LPLI arecoline-induced OSF. Arecoline stimulated human gingival fibroblasts (HGFs) were treated with or without LPLI. The expression of fibrotic marker genes, α-SMA and CTGF, were analyzed by quantitative real-time RT-PCR and Western blots. The transcriptional activity of CTGF was further determined by the reporter assay. Our data showed that arecoline increased the mRNA and protein expressions of α-SMA and CTGF in HGF. Interestingly, both LPLI and forskolin , an adenylyl cyclase activator, reduced the expressions of arecoline-mediated fibrotic marker genes and inhibited the transcription activity of CTGF. Moreover, pretreatment of SQ22536, an adenylyl cyclase inhibitor, blocked the LPLI’s inhibitory effect of the expressions of arecoline-mediated fibrotic marker genes. Our data suggest that LPLI may have anti-fibrotic effects via the cAMP signaling pathway.