| Abstract: | 口腔黏膜下纖維化症(oral submucous fibrosis；OSF)為一種口腔癌前期病變，主要與檳榔的嚼食習慣有關。而造成口腔黏膜下纖維化症的主要原因是細胞外基質中膠原蛋白異常的堆積及分解所導致的。基質金屬蛋白水解 (matrix metalloproteinases；MMPs)及纖維蛋白溶解系統(plasminogen activator (PA)/plasmin system)在細胞外基質的組成及分解過程中扮演很重要的角色。可是 MMPs、t-PA 及 PAI-1 在口腔黏膜下纖維化症致病過程中的機制卻仍不清楚。因此，我們除了觀察上述基因在正常頰黏膜及口腔黏膜下纖維化的差異性表現之外，也進一步利用檳榔素(arecoline)來探討其引發口腔黏膜下纖維化症的致病機轉，試圖解釋由嚼食檳榔所引發之口腔黏膜下纖維化症可能的原因。因此我們利用 gelatin zymography、western blotting 及 ELISA 等分析方式，發現在口腔黏膜下纖維化症的纖維母細胞比正常頰黏膜的纖維母細胞有較高的 TIMP-1、t-PA 及 PAI-1的蛋白表現，而在 PAI-1 的 mRNA 轉錄層次也有顯著的上升。我們也進一步利用檳榔素來探討其引起口腔黏膜下纖維化症的致病機轉。由實驗結果發現，當正常頰黏膜的纖維母細胞在處理不同濃度的檳榔素(10, 20, 40, 80 ug/ml)之後， TIMP-1、t-PA 及 PAI-1 的蛋白表現都有顯著的上升，而 MMP-2 的蛋白表現則隨著檳榔素濃度的增加而有明顯的降低。另外，我們也觀察 PAI-1 promoter region 第-675bp位置的4G/5G 基因多型性。我們利用 ASPCR 及 ASRS 的方式證明 PAI-1 在正常頰黏膜組織中的 4G/4G : 4G/5G : 5G/5G 的比例為 21.9% : 46.9% : 31.2 %；而在口腔黏膜下纖維化組織中的4G/4G : 4G/5G : 5G/5G 的比例為 42.3% : 44.2% : 13.5 %，而我們進一步用卡氏平方的統計方式發現在正常頰黏膜組織及口腔黏膜下纖維化的組織中，其 PAI-1 的 4G/5G 基因多型性具有統計學上的意義(P<0.05)。根據上述結果可以發現，形成口腔黏膜下纖維化症的可能原因是由於 MMP-2 的減少及 TIMP-1 的增加，而導致細胞外基質中膠原蛋白異常的堆積及分解。而在 PAI-1 方面，由於口腔黏膜下纖維化症患者中 4G/4G 的比例明顯增加，可能進而導致 PAI-1 蛋白表現的增加，而增加了口腔黏膜下纖維化症形成的機率。
Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in areca quid chewers. It is probably a consequence of disturbances in the hemeostatic equilibrium between synthesis and degradation of extracellular matrix molecules (ECM). To date, there has been little research about the role of matrix metalloproteinases (MMPs) and plasminogen activator (PA)/plasmin system in the pathogenesis of OSF. In the present study, we examined the activity of TIMP-1 and PAI-1 from cell cultured from OSF and normal buccal mucosa. OSF specimens were found to have higher TIMP-1 and PAI-1 expression than normal buccal mucosal fibroblasts (BMFs) by Western blots. To verify whether arecoline, a major areca nut alkaloid, could affect TIMP-1 or MMP-2 production by human BMFs, Western blots and gelatine zymography were used. Arecoline was found to elevate TIMP-1 and PAI-1 expression at the concentration level under 20 ug/ml in a dose-dependent manner. From gelatin zymograms, the main gelatinolytic proteinase secreted by the human BMFs was MMP-2, and only minimal amounts of MMP-9 could be detectable from zymogram. In addition, arecoline was found to inhibit MMP-2 secretion and production in a dose-dependent manner. In this study, we also investigated the genetic analysis of PAI-1 in the promoter region between OSF and normal buccal mucosa. PAI-1 genotyping with allele specific polymerase chain reaction (ASPCR) and allele-specific restriction enzyme site analysis (ASRS) was performed in the tissue of 52 OSF and 32 normal buccal mucosa. There were significant differences between the OSF and BMF for the frequencies of the 4G/4G, 4G/5G and 5G/5G genotypes (P < 0.05). In the OSF group, it had a hight frequency of PAI-1 (4G/4G) genotypes than those in BMF group (P < 0.05) Taken together, it was found that arecoline acted not only as an inhibitor on gelatinolytic activity of MMP-2, but also a stimulator for TIMP-1、t-PA and PAI-1 activity. These synergistic effects may contribute to the ECM components accumulation in the areca quid associated OSF. Furthermore, our finding also suggested that the distribution pattern of PAI-1 promoter were different between OSF and BMF tissue. PAI-1 4G allele, with a higher transcription activity, was more prevalent in OSF. |
