口腔黏膜下纖維化症中B7-H1表現之研究

Abstract

口腔黏膜下纖維化症(Oral submucous fibrosis，OSF)是一種持續性發炎所導致的口腔黏膜病變，屬於潛在性癌前障礙，主要因口腔黏膜下組織層過多的膠原蛋白堆積，而造成纖維化。已知在發炎的過程中，部分發炎前細胞激素會被大量分泌，其中像是IFN-γ、TGF-β等，此些激素參與纖維化的致病機轉已被進一步研究，另外，在之前的文獻中指出，B7-H1蛋白表現會受到TGF-β、IFN-γ、TNF-α等細胞激素所調控，因此，在本研究中，其主要目的是在探討B7-H1於口腔黏膜下纖維化症之致病機轉中，其所扮演的角色。 首先，利用B7-H1抗體進行免疫組織化學染色，偵測在正常口腔黏膜組織(normal oral mucosa；NOM)及口腔黏膜下纖維化症組織(oral submucosa fibroblast；OSF)的切片中B7-H1表現的情形，結果發現，在OSF的切片中明顯看到部分表現B7-H1的纖維母細胞靠近上皮層組織，且同時觀察到在上皮組織層中B7-H1的表現也較正常組織中多。其次，正常黏膜纖維母細胞以arecoline處理後，細胞中B7-H1表現呈現增加的趨勢，並且發現用JNK抑制劑可抑制B7-H1的表現。過去文獻指出在人類皮膚癌中，B7-H1的過度表現會去調控上皮-間質轉換現象(EMT)，而本研究免疫組織化學染色結果也發現，在OSF組織中，表現B7-H1的纖維母細胞大多集中在上皮下結締組織，推測可能有EMT現象產生而導致纖維母細胞的增生活化。利用細胞實驗看in vitro中B7-H1表現，用TGF-β去處理S-G上皮細胞，發現在S-G細胞中EMT 生物標記蛋白增加且B7-H1表現也增加，處理JNK抑制劑可以抑制B7-H1的表現，並且發現EMT biomarker蛋白也有減少的趨勢，進一步用B7-H1 shRNA來確認B7-H1是否會進一步去調控EMT，結果顯示當抑制B7-H1表現時，會影響EMT相關蛋白的表現。

Oral submucous fibrosis is a continuous, chronic, insidious and inflammatory disease of oral mucous that is a kind of potentially malignant disorders. OSF is mainly due to consumed areca quid which is lead to fibrosis in the oral cavity. In the progression of inflammatory, some pro-inflammatory and pro-fibrotic cytokine could be upregulated, like TGF-β、IFN-γ, etc., the exact pathogenesis of OSF and cytokine was still unclear and needed to study .However, the previously study had showed that B7-H1 was regulated by IFN-γ in the human dermal fibroblast. Therefore, in this study, we investigated whether B7-H1 was involved in the mechanisms of OSF diseases. First, we investigated the expression of B7-H1 in normal oral mucosa tissue and in OSF patient’s tissue by immunohistochemistry (IHC).The result showed that most of fibroblasts that express B7-H1 was close to epithelium layer in OSF tissue. It also showed that the expression of B7-H1 in epithelium layer in OSF is much more than in NOM. Then, we treated NOM fibroblast with arecoline and analyzed the expression of B7-H1 by Western blot. The result showed that B7-H1 is up-regulated by arecoline in NOM, and JNK inhibitor could reduce B7-H1 expression. The reference indicated that B7-H1 over-expression could be regulated Epithelial-mesenchymal transition (EMT) in human skin cancer. The result of IHC showed that fibroblasts that express B7-H1 were in the juxta-epithelial connective tissue. The reference showed that EMT is one of important mechanisms of OSF, and to investigate whether B7-H1 is involved in EMT which is lead to fibrosis. We treated S-G epithelial cell with TGF-β, and the result showed that EMT biomarker protein and B7-H1 expression was increased. And then, we pre-treat S-G cell with JNK inhibitor and then treat with TGF-β, the result showed that the expression of B7-H1 and some EMT biomarker both were reduced. And then, we used B7-H1 specific siRNA to knockdown its expression in S-G cells. The result showed that knockdown of expression of B7-H1 influenced EMT biomarker gene expression.