口腔黏膜下纖維化症之致病基因微陣列分析及蛋白質確認

The Micro-Array Analysis of Genetic Change and Proteins Identification of Oral Submucous Fibrosis

高文斌(Wen-Bin Kao) ； 謝耀東(Yao-Dung Shieh) ； 謝天渝(Tien-Yu Shieh)

《臺灣口腔顎面外科學會雜誌》 19卷4期 (2008/12) Pp. 249-259

https://doi.org/10.7104/TJOMS.200812.0249

摘要

口腔黏膜下纖維化症（簡稱OSF）是一種慢性、纖維化、不可逆性的癌前病變。病因尚未明確，但被視爲口腔癌癌前病變。本研究的目的是從基因微陣列分析(micro-array alanysis)、免疫組織化學染色、反轉錄聚合酶鏈鎖反應(polymerase chain reaction)等方式來比較正常口腔黏膜與口腔纖維化黏膜的篡因表現有那些差異。結果顯示有1316個基因顯現有差異，其中有18個基因表現(gene expression)上升，10個基因表現下降，另外還有6個house keeping基因。在18個基因表現上升的基因群中，鱗狀上皮癌抗原第一型(squamous cell carcinoma antigen 1)的發炎反應較明顯。用免疫組織化學染色來分析特定蛋白質表現之結果，並利用獨立樣本t檢定(independent-samples t-test)來分析，發現不管是Co1 Ⅰ、Co1 Ⅲ、PDK或P450 reductase等蛋白質在OSF及正常人的黏膜上皮層或結締組織層的細胞中，染色反應並與統計上的明顯差異；但是不管在OSF或正常人的結締組織層細胞中，Co1 Ⅲ的染色反應都比CoⅠ強，且具有統計上的意義。在細胞色素P450還原酶的免疫化學染色結果方面，雖然發現在OSF患者口腔黏膜的患側比正常側染色結果較弱，但是沒有統計上的意義(P=0.015)。然而從定量即時反轉錄聚合酶鏈鎖反應的結果來看，發現OSF患者在其患側口腔黏膜的P450基因表現比正常人少4倍(Ratio=0.26)；甚至OSF患者本身的正常側的口腔黏膜其P450基因表現也比正常人口腔黏膜組織P450的基因表現要少2倍(Ratio=0.55)。因此本研究結果顯示口腔黏膜下纖纖化患者與正常人的口腔黏膜基因表現中，以細胞色素P450還原酶方面的差異最明顯，或許這個差異與形成口腔黏膜下纖維化的機轉有重要關係，或許也可提供後續口腔黏膜下纖維化病變相關研究之思考方向。

關鍵字

口腔黏膜下纖維化 ； 反轉錄眾合酶鏈鎖反應 ； 免疫組織化學染色

並列摘要

Oral submucous fibrosis (OSF) is an irreversible, chronic, fibrotic change of oral mucosa. It is also known as a premalignant oral lesion. The purposes of our study are to use micro- array analysis, immunohistochemical staining, and polymerase chain reaction methods to compare the gene expression profile of oral submucous fibrosis tissue with the normal oral mucosa tissue. 54 cases of normal oral tissue and 72 cases of clinically diagnosed OSF with pathological evidence were included in this study. The results revealed significant change in 1316 genes, among them 18 genes being up-regulated, 10 genes being down- regulated and 6 genes being house keeping. The results from immunohistochemical staining show that there is no statistical difference in Co Ⅰ, Co Ⅲ, PDK and P450 reductase gene expression between the OSF tissue and normal oral tissue. However, the gene expression of Co Ⅲ is stronger than Co Ⅰ in either OSF or normal oral tissue. In OSF group, P450 reductase gene expression at the fibrotic tissue is stronger than non-fibrotic oral tissue but no statistical difference (p=0.015). The results from quantitative real-time PCR show that the expression of P450 mRNA in the fibrotic tissue of OSF is 4 times less than that in the normal oral tissue. Compared P450 mRNA expression in non-fibrotic tissue of OSF patients with normal oral tissue, the P450 mRNA expression is 2 times less in the non-fibrotic tissue of OSF patients. We further compared the expression difference of Col Ⅰ, Col Ⅲ, PDK and P450 in the fibrotic tissue and non-fibrotic tissue of same OSF patients. By use of Paired-Samples t-test analysis, the results from the P450 gene expression revealed no statistically significant difference (P=0.015). The non-fibrotic oral tissues had mean 2.40±SD 0.52 while the fibrotic oral tissues had mean 1.90±SD 0.32. These findings in the P450 mRNA expression of OSF patients probably provided genes expression profile and protein identification which may be associated with OSF pathogenesis. They, therefore, may be used in the future to do further OSF researches.

並列關鍵字

Oral Submucous Fibrosis ； Real-time PCR ； Immunohistochemistry Staining