應用微矩陣生物晶片探究檳榔素對人類口腔齒齦纖維母細胞之基因表現

Application of cDNA Microarray Technique for Gene Expression Analysis in Arecoline treated Human Oral Gingival Fibroblasts

王佾柔(Yi-Jou Wang)

指導教授 ： 葛應欽

高雄醫學大學/口腔醫學院/口腔衛生科學研究所碩士班/碩士(2005年)

摘要

研究背景：
國際癌症研究中心在2003年宣佈，單獨嚼食檳榔塊(不含菸草)，對人類致癌證據充分，為第一類致癌物，而「檳榔果」也新認定為第一類致癌物。許多研究亦證實口腔癌及口腔黏膜下纖維化的發生與嚼食檳榔有明顯的相關性。而檳榔素為檳榔中主要的化學物質，然而關於此成分的代謝機制及致癌機轉仍不甚明瞭。
研究目的：
本研究希望藉著實驗室培養正常人類齒齦纖維母細胞株，以點製有46000個人類基因的cDNA基因晶片，來大量篩檢所表現出來的代謝酵素或其他重要相關基因，並探索這些基因與嚼食檳榔之間致病的關聯性。另外，在微陣列資料分析部份，運用Empirical Bayesian、Student’s t-test、SOM及K-means之不同統計分析方式，交叉比對後選出程度最強之表現差異顯著的基因。
研究方法：
實驗室培養正常人類齒齦纖維母細胞株(HGF-1)繼代培養至第四代，其中實驗樣本(5個)為投以檳榔素濃度100μg/ml刺激作用之細胞，而以不加檳榔素之樣本為對照組(1個)，經培養箱培養24小時後，分別萃取細胞之RNA，再以點製有46000個人類基因的cDNA基因晶片，來大量篩檢所表現出來的代謝酵素或其他重要相關基因。
結果：
結果顯示，有58個基因表現有上升的現象，另外有606個基因表現下降。這些表現上升或下降的基因根據SOURCE Search網站所提供的資料庫比對顯示，功能歸類為細胞骨架、轉錄因子、代謝、細胞週期、轉譯、細胞凋亡、抑癌基因等。其中可比對功能的基因大多有關細胞中傳導物質的訊息表現，而有關檳榔素之可能代謝基因為prostaglandin-endoperoxide synthase 2 (COX-2)，此為代謝的活化基因，而其他亦有許多的基因參與了口腔細胞纖維化之過程，其中有10個基因和膠原蛋白有關；另外有1個基因為MMP家族的基因，另外也發現有5個癌化基因的參與，但其基因分型，卻為先前檳榔相關研究中所沒提及過的。
結論：
本研究屬於初步探索性型的研究，初步的結果發現相當廣泛，在未來的研究，緊接著是以RT-qPCR及蛋白質體學的技術作為驗證平台。也相信隨著基因體資訊的漸趨成熟後，這種微矩陣基因晶片研究方式應該可以提供更多的方向去探討目前致癌機轉不明的致癌物，相信對口腔癌症的防治有絕對的幫助。

關鍵字

微矩陣生物晶片 ； 口腔齒齦纖維母細胞 ； 檳榔素

並列摘要

Background:
By the year 2003, the IARC has declared that chewing of betel quid, by itself, to be a Group 1 carcinogen and the areca nut to be, correspondingly, a Group 1 carcinogen. Many epidemiological studies also indicated that Betel quid chewing has a strong association to the incidence of oral cancer and oral submucous fibrosis. Arecoline is the major chemical element of betel nuts, while the mechanism of carcinogenesis or metabolism associated with this compound is not well understood.

Study objective:
Five repeated pairs were hybridized on 4.6K cDNA chip respectively to perform the association between areca nut chewing and expressive genes. Further, the significantly differential expressive genes were screened and performed by using Empirical Bayesian method, Student’s t-test, SOM, and K-means clustering synchronously.

Methods:
In this study, the normal human HGF-1 cell lines were treated with 100μg/mL arecoline and maintained at incubator 24 hours as case samples, comparing to control samples without treated, before total RNA extraction. Five repeated pairs were hybridized on 4.6K cDNA chip respectively to perform the association between areca nut chewing and expressive genes.

Results:
The results indicate that there are 58 up-regulated genes and 606 down-regulated genes in the research. The performances of those genes, based on comparing statistics provided by the web site of the SOURCE Search, can be categorized into cytokines, transcription factor, metabolism, cell cycle, translation, apoptosis, tumor suppressor genes. Moreover, those genes that can be compared exhibit trace of signal transduction, and among those genes, prostaglandin-endoperoxide synthase 2 (COX-2) is a possible metabolic activation gene that is highly related to Arecoline. In addition, other genes are also observed to take part in oral cell fibrosis among which 10 of them are correlated with collagen and one of them comes from MMP superfamilly. Finally, five carcinogenetic genes whose genotype were never found and observed in previous related research contribute to oral fibrosis as well.

Conclusion:
This is a preliminary research whose findings are basic and general. Future studies will be using RT-qPCR and proteomics as a test platform. Furthermore, with the development and maturity of Genome information, the current research based on microarray can provide more insights to carcinogenetic mechenism, which will greatly contribute to the prevention of oral cancer.

並列關鍵字

arecoline ； oral gingival fibroblasts ； cDNA microarray

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