口腔黏膜下纖維化症之特定基因及其蛋白質表現分析

背景：口腔黏膜下纖維化症是一種慢性、纖維化、不可逆性的癌前病變，一般認為嚼食檳榔是造成口腔黏膜下纖維化症(Oral Submucous Fibrosis, OSF)之重要因素，但有些人、有些種族長期嚼食檳榔卻不會得到OSF，所以個體基因型態與是否會患OSF是息息相關的。研究目的：當人類基因體序列漸漸被解讀後，我們利用「基因晶片微陣列分析」之輔助，來研究口腔黏膜下纖維化之患者的基因是否具有某些特異性。研究方法：本研究收集國軍高雄總醫院口腔顎面外科2001年9月至2003年8月間門診及住院口腔黏膜下纖維化之患者，這些患者有張口受限、口腔黏膜瓣、口腔黏膜變白、口腔黏膜僵硬等症狀共計72位，另收集正常組織標本做為控制組，該類組織係由門診中拔除下顎阻生第三大臼齒的病患，取得其黏膜組織共計54位。研究設計：收集口腔黏膜下纖維化之患者及正常人組織進行H-E stain 染色、觀察組織形態，抽取組織的核醣核酸(RNA)，反轉錄成互補去氧核醣核酸(cDNA)進行微陣列雜交，經資料的掃瞄與分析，確認並選取變異較大的基因。將該基因對應蛋白質之抗體，用免疫組織化學染色法(Immunohistochemistry, IHC)來染色、觀察、紀錄及分析，並應用SPSS軟體的獨立樣本t檢定及相依樣本t檢定進行統計分析，再運用反轉錄聚合酶鏈鎖反應(RT-PCR)來印證所研究基因的表現狀態。結果：經基因晶片微陣列分析偵測出有18個基因表現上升，10個基因表現下降，共同高度表現的基因有1,316個，其中有6個是house keeping genes，從口腔黏膜下纖維化之患者表現上升的基因群裡，我們選擇mRNA蛋白酶第一型（3-phosphoinositide-dependent protein kinase-Ｉ, PDK）、細胞色素氧化還原酶（cytochrome P450 reductase, P450），及已知與口腔黏膜下纖維化組織結構息息相關的膠原蛋白第一型(CollagenＩ, ColＩ)、膠原蛋白第三型(CollagenⅢ, ColⅢ)進行免疫組織化學染色分析，經獨立樣本t檢定統計，得知口腔黏膜下纖維化之患者與正常人的口腔黏膜組織雖然在基因有表現強度的差異，但經免疫組織化學染色，不管是PDK、P450、 Col Ⅰ或Col Ⅲ，在口腔黏膜下纖維化之患者及正常人的口腔黏膜上皮層或結締組織層的細胞中，染色反應並無統計上的明顯差異。我們使用口腔黏膜下纖維化同一患者的患側及正常側的口腔黏膜進行免疫組織化學染色統計分析，經統計方法相依樣本t檢定，結果發現P450在結締組織層中，患側(1.90±0.32)比正常側(2.40±0.52)明顯較少，在統計上有明顯差異（P=0.015）；再經反轉錄聚合酶鏈鎖反應(RT-PCR），結果發現P450還原酶在口腔黏膜下纖維化患者之患側的mRNA(Ratio＝1.95)比正常人(Ratio＝7.49)少4倍，甚至在口腔黏膜下纖維化之患者的正常側(Ratio＝4.47)黏膜組織其P450還原酶的基因表現比正常人的黏膜組織少2倍。結論描述：本研究發現口腔黏膜下纖維化之患者與正常人表現差異的基因有SCC Ag. Retinoblastoma binding protein 3, Cytochrome P450 reductase等，其功能與細胞凋亡、細胞增生、細胞週期、轉錄因子.... 等均有密切關係，由此確認特定之基因如P450 ，在OSF的病程中佔有重要之角色，亦是吾人未來要研究此疾病的一個標的。

關鍵字

免疫組織化學染色；口腔黏膜下纖維化；反轉錄聚合?鏈鎖反應

並列摘要

Background： The pathogenesis of oral submucous fibrosis(OSF) is unknown in medical reports. We try to find out those genes correlated with OSF. To explain the OSF disease was induced not only by betel by nut chewing but also by personal genes factors. Study objective： OSF is a chronic inflammatory and irreversed precancerous oral lesion. The etiologic factors in OSF are variant. We compared the genes and proteinsexpression profile between the normal oral tissues and OSF tissues by using cDNA micro-array and immunohistochemistry stainning to analysis the genetic change and protein identification respectively. Methods： Fifty four case of normal tissues from extracted mandibular third molar were included in this study as control group. Seventy two cases of clinical diagnosed OSF with evidence of histopathological proof and mouth open limitation with fibrosis band were included in this study as experimental group. Fresh tissues biopsy of oral mucosa were taken from the Department of Oral and Maxillofacial Surgery of Kaohsiung Military General Hospital. We used H-E stain method to differenciate the normal mucosa and OSF mucosa. The microarray analysis of genetic change and immunohistochemistry(IHC) stainning method were used to identify these parculiar proteins between the normal oral tissues and OSF tissues. After IHC stainning examination, we used Reverse Transcriptase Polymerase Chain Reaction(RT-PCR) to identify the expression pattern of those genes. Results： The results revealed significant change in 1316 genes, with 18 genes being up-regulated, 10 genes being down-regulated and 6 genes being house keeping. The list of these genes including squamous cell carcinoma antigen I, P450 etc. they function as cell cycle, cell proliferation, cell apoptisis, protein metabolism, redox enzyme, transport factor and inflammation. From Independent-samples t Test analysis the Col I & Col III over OSF patient’s connective tissues had a statistically significant difference(P=0.000), Col III intensity 2.90±0.30 is higher than that of Col I 1.05±0.74. For same OSF patient, we compared the lesion side and normal side using the antibody Col I, Col III, PDK and P450. The results revealed a statistically significant difference(P=0.015) in P450 antibody among normal side connective tissues 2.40±0.52 and lesion side connective tissues 1.90±0.32. From the RT-PCR results, we compared the genes expression profile between the same OSF patient lesion side mucosa and normal side mucosa and normal tissues mucosa. We found the OSF lesion(Ratio＝1.95) side P450 genes expression 4 times lesser than normal tissues(Ratio＝7.49) and normal side(Ratio＝4.47) OSF genes expression 2 times lesser than for normal tissues. Conclusion： From micro-array analysis , IHC stainning, RT-PCR showed that results PDK、P450 had no significant difference in OSF and normal tissues mucosa, even they had high expression of micro-array genes analysis. These results provided a large amount of information regarding genes expression profiled which is associated with OSF. The no consistency expression in IHC stainning and RT-PCR may resulted from change in transcription and translation process genes expression profiled and proteins identification provided diagnostic, preventive and therapeutic utilization in future study of OSF.