檳榔塊成分對人類口腔粘膜纖維化及癌化之影響

Effects of betel quid ingredients on human oral mucosal fibrosis and carcinogenesis

謝典華(Dean-Hwa Shieh)

指導教授 ： 謝天渝 ； 蔣連財

高雄醫學大學/口腔醫學院/牙醫學研究所/博士(2003年)

摘要

(oral submucous fibrosis，OSF)主要是膠原蛋白異常堆積所造成，為一種口腔癌前期病變，主要與嚼檳榔的習慣有關，台灣嚼檳榔的人口超過二百萬，其中嚼檳榔又有抽菸者約86%，嚼檳榔的方式各地不同，流行病學的研究顯示嚼荖花檳榔塊比荖葉檳榔塊有較高的口腔癌發生率。檳榔的化學組成中，含量最多的是檳榔素，其具有細胞毒性及基因毒性，會抑制細胞附著、擴張、移動、生長且會抑制細胞間質蛋白及DNA合成，檳榔素會水解成檳榔次鹼；荖花的成份中，含量最多的是黃樟素，為已知的致癌物，也具有細胞毒性及基因毒性，會抑制細胞DNA合成；至於菸的內含物中，尼古丁會抑制細胞的生長及附著，且會增強檳榔素的細胞毒性。
本研究主要是探討檳榔塊成分對人類口腔粘膜纖維化及癌化之影響，內容包括以下六大部分：一、正常人和口腔粘膜纖維化症患者纖維母細胞分泌TIMP-1差異性研究。二、檳榔塊成分對同一個口腔粘膜纖維化患者的正常部位及纖維化部位之口腔粘膜纖維母細胞及上皮細胞存活性的影響。三、檳榔塊成分對同一個口腔粘膜纖維化患者的正常部位及纖維化部位之纖維母細胞TIMP-1基因表現的影響。四、檳榔塊成分對同一個口腔粘膜纖維化症患者的正常及纖維化部位纖維母細胞之膠原蛋白吞噬作用的影響。五、檳榔塊成分對口腔粘膜纖維母細胞及上皮細胞在生體外關於口腔癌癌化機轉之探討。六、口腔癌癌化過程致癌及抑癌基因表現之探討。
關於口腔粘膜纖維化方面，來自不同人的纖維母細胞，OSF與正常纖維母細胞的TIMP-1蛋白分泌量並沒有顯著的差異性，且TIMP-1蛋白與性別、年齡、抽菸、喝酒、嚼檳榔等因子沒有顯著的相關性，而危險因子與口腔粘膜下纖維化病變間的相關性大小，依序為檳榔＞菸＞酒。
由於來自不同個體的樣本可能因先天基因的易感性不同，而有個體差異性存在，因此本研究使用來自同一個人正常及OSF部位的纖維母細胞來做研究發現：同一試劑濃度刺激後，同一個人其OSF部位及正常部位所培養的口腔粘膜纖維母細胞及上皮細胞數目並沒有明顯的不同；而大於50µg/ml的檳榔素或黃樟素及大於100µg/ml的尼古丁對OSF部位或正常部位口腔粘膜纖維母細胞的生長都有抑制作用，且隨著濃度的增加，細胞生長抑制作用更加明顯，檳榔次鹼則有促進細胞生長作用。大於25µg/ml的檳榔素或黃樟素對OSF部位或正常部位口腔粘膜上皮細胞的生長都有抑制作用
同一個人的正常與OSF部位的組織所培養的纖維母細胞分泌TIMP-1蛋白及mRNA表現有顯著的差異：OSF部位的纖維母細胞產生的TIMP-1蛋白及mRNA皆高於正常部位；大於10µg/ml的檳榔素會促使正常與OSF部位纖維母細胞分泌的TIMP-1蛋白及mRNA有顯著的上昇；大於10µg/ml的黃樟素會促使正常部位纖維母細胞分泌的TIMP-1蛋白增加，而大於25µg/ml的黃樟素會促使正常與OSF部位纖維母細胞分泌的TIMP-1蛋白增加，且mRNA也有顯著的上昇。
分析來自同一個人的纖維母細胞，其膠原蛋白吞噬能力的差異，結果發現：正常部位組織所培養的纖維母細胞其含有膠原蛋白吞噬作用的細胞數量較OSF部位為多；大於10µg/ml的檳榔素會使正常部位有吞噬作用的纖維母細胞顯著減少，大於25µg/ml的檳榔素會使OSF部位有吞噬作用的纖維母細胞顯著減少；大於25µg/ml的黃樟素會使正常及OSF部位有吞噬作用的纖維母細胞顯著減少；大於50µg/ml的尼古丁會使正常及OSF部位有吞噬作用的纖維母細胞顯著減少。而25µg/ml的尼古丁對於檳榔素或黃樟素所造成的吞噬細胞數量減少有加成作用；由以上的研究結果得知：受到不同檳榔塊成分刺激後纖維母細胞的TIMP-1表現增加，吞噬作用降低，可能促進了口腔粘膜纖維化症的發生。
至於檳榔成分與口腔癌癌化的相關性方面，利用培養的口腔粘膜上皮細胞及纖維母細胞以檳榔成分刺激，觀察一些腫瘤標記的變化，結果發現：未受檳榔成分刺激時OSF細胞各種tumor marker表現量大於正常細胞，印證了OSF為口腔癌前期病變；OSF上皮細胞cytokeratin 19濃度與正常上皮細胞有顯著差異，且隨著arecoline及safrole濃度的增加其差異更明顯；OSF上皮細胞telomerase量顯著高於正常上皮細胞，OSF纖維母細胞telomerase量也顯著高於正常纖維母細胞，而且受arecoline及 safrole刺激時telomerase量也有顯著的增加；OSF上皮細胞及纖維母細胞DNA aneuploidy與正常上皮細胞及纖維母細胞無顯著差異。檳榔塊不同成分刺激後誘生tumor marker的情況不同，由此推論癌化的過程是多基因所控制且多步驟逐漸進行的事實。細胞受到檳榔塊成分刺激後會產生何種tumor marker的變化，是有個體差異的，這也說明了個體癌化的易感性可能不同。根據前述結果推論由口腔粘膜纖維化症轉變成口腔癌的可能機制為：上皮細胞受到檳榔塊成分的刺激起變化，同時纖維母細胞也受刺激分泌某些物質來促進上皮細胞逐漸癌化。
使用DNA microarray來探討口腔癌癌化過程可能的致癌及抑癌基因變化，由研究結果推論：在口腔癌癌化過程中，由正常組織轉變成OSF病變時，可能有表現異常的基因為c-fos、c-myc、E-cadherin；由OSF病變再轉變成口腔癌時，可能有表現異常的基因為EGFR、erbB-2、c-myc、p53，而這些基因的變化有待進一步的研究驗證。

關鍵字

口腔粘膜下纖維化病變 ； 口腔癌 ； 檳榔素 ； 檳榔次鹼 ； 黃樟素 ； 尼古丁 ； 金屬蛋白分解酵素組織抑制劑 ； 膠原蛋白吞噬作用

並列摘要

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by excessive deposition of fibrous contents in submucous layer of oral connective tissues that leads to limitation of mouth opening and increasing difficulty in speaking, chewing and swallowing. It results from an increased collagen synthesis, and a decreased collagenase activity. Collagen degradation is demonstrated via the intracellular phagocytosis route and by the extracellular collagenase-dependent route. The extracellular regulation involves the balance of matrix metalloproteinases and tissue inhibitor of metalloproteinases (TIMPs). Elevated TIMP-1 protein has been thought to be associated with oral fibrosis, however whether TIMP-1 expression in OSF is modulated at the transcriptional level is still unknown. OSF is an oral precancerous condition, and is associated with betel quid (BQ) chewing habits. The present study used arecoline, arecaidine and safrole, which were thought to be major toxic ingredients in BQ, as candidates to explore the role of TIMP-1 expression in OSF pathogenesis. The results indicated that TIMP-1 protein secretion by OSF fibroblasts was more than normal fibroblasts from the same patients, and the mRNA expression of TIMP-1 in OSF fibroblasts was higher than normal fibroblasts of the same patients. Arecoline and safrole significantly elevated TIMP-1 protein and mRNA expression.
In Taiwan, 86% of betel quid chewers are also smokers. This study also used arecoline, safrole and nicotine, which are released in saliva during BQ chewing plus cigarette smoking, to evaluate the collagen phagocytosis by fibroblasts. The results demonstrated that collagen phagocytosis by fibroblasts from the normal region was higher than from the OSF region of the same patients. Collagen phagocytosis by fibroblasts exhibited a dose-dependent inhibition as the concentration of arecoline, safrole and nicotine increased. Besides, nicotine had a synergistic effect on arecoline- or safrole-inhibited collagen phagocytosis. Our results concluded that heterogeneity of fibroblasts existed in the same patients and increased mRNA expression of TIMP-1 in buccal mucosal fibroblasts by arecoline and safrole as a possible mechanism for oral submucous fibrosis. In addition, deficiency in collagen phagocytosis of fibroblasts might participate in the pathogenesis of oral submucous fibrosis. Arecoline, safrole and nicotine inhibit collagen phagocytosis by fibroblasts may induce OSF formation in Taiwan's patients.
On the other hand, oral submucous fibrosis could progress to oral cancer, but the mechanism of malignant change was still unknown. The research would explore this oral carcinogenesis mechanism. Added different concentrations of arecoline and safrole to the cultured oral epithelial cells and fibroblasts, and detected the telomerase activity and DNA aneuploidy. Besides, this study also quantified the cytokeratin 19 levels of oral epithelial cells. The results depicted that arecoline and safrole inhibited the proliferation of oral epithelial cells and fibroblasts. There were significant differences in cytokeratin 19 levels between normal and OSF epithelial cells of the same patients, and the cytokeratin 19 levels augmented as arecoline and safrole concentrations increased. Telomerase activity was significant difference between normal and OSF fibroblasts of the same patients, and was significant difference between normal and OSF epithelial cells of the same patients. Arecoline and safrole showed to elevate telomerase activity. No significant difference of DNA aneuploidy was observed between normal and OSF cells of the same patients. Our results concluded that arecoline and safrole could induce tumor marker expression, and there might be individual variations. The possible carcinogenesis mechanism of OSF to oral cancer was that the oral epithelial cells and fibroblasts were stimulated by the betel quid contents at the same time, and oral fibroblasts secreted some substances to promote the malignant change of the oral epithelial cells.
The present study also observed the expression of oncogenes and tumor supressor genes in oral carcinogensis. We collected the tissue specimens from normal oral mucosa, oral submucous fibrosis lesion and oral squamous cell carcinoma lesion of the same patients, and measured oncogene or tumor supressor gene expression by using DNA microarray. The results indicated that changes in gene expression of c-fos, c-myc and E-cadherin were the early events in oral carcinogenesis and followed by the changes in gene expression of EGFR, erbB-2, c-myc and p53. These findings need further issues to clarify the correlation between the changes of these genes and oral carcinogenesis.

並列關鍵字

oral submucous fibrosis ； oral cancer ； arecoline ； arecaidine ； safrole ； nicotine ； tissue inhibitor of metalloproteinases ； collagen phagocytosis

參考文獻

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