新型口腔黏膜白斑惡變細胞模型的建立及惡變相關(guān)差異基因組檢測的研究
發(fā)布時間：2018-08-30 09:34
【摘要】：口腔鱗狀細胞癌(Oral squamous cell carcinoma,OSCC)是一種常見的頭頸部惡性腫瘤,占口腔惡性腫瘤80%以上,容易復(fù)發(fā)和轉(zhuǎn)移,預(yù)后較差,5年存活率約50%,且近年來有上升和年輕化的趨勢,是一種嚴重威脅人類健康的疾病,越來越引起人們的重視,但該病的發(fā)生發(fā)展分子機制目前尚不完全清楚。口腔鱗狀細胞癌常常起源于口腔黏膜的癌前病變,尤其是口腔黏膜白斑(Oral leukoplakia,OLK),口腔黏膜白斑常因受到煙草等物質(zhì)的刺激而導致癌變,而煙草中的關(guān)鍵致癌物質(zhì)是苯并芘(Benzo(a)pyrene,Bap)和二甲基苯并蒽(7,12-dimethyl-benz[a]anthracene,DMBA),在本次研究中,我們首次建立了由B(a)P和DMBA的聯(lián)合誘導輕中度異常增生的口腔黏膜白斑組織細胞(Dysplastic oral keranticyte,DOK)惡變的新的口腔鱗狀細胞癌細胞模型,命名為OSCC-BD細胞;通過對口腔黏膜白斑細胞DOK和其惡變后的細胞OSCC-BD的細胞形態(tài),細胞周期,生長能力和體外侵襲能力等進行研究,從而證實OSCC-BD細胞的惡性特征。盡管早期診斷和治療對于口腔鱗狀細胞癌的預(yù)后非常重要,但是其致病機制及相關(guān)的有效分子標志物仍舊缺乏,本實驗通過基因芯片技術(shù)對已建立的口腔黏膜白斑惡變前后的細胞進行差異基因組的研究,以尋找能預(yù)測口腔黏膜白斑惡變的有效分子標志物,對口腔黏膜白斑惡變的早期發(fā)現(xiàn)、早期診斷、早期治療及改善患者預(yù)后,預(yù)防口腔鱗狀細胞癌的發(fā)生具有重要的科學和臨床意義。目的:通過B(a)P和DMBA合劑誘導口腔黏膜白斑細胞DOK惡變,建立新的口腔黏膜白斑惡變的細胞模型即OSCC-BD細胞系,并用基因芯片技術(shù)分析檢測惡變相關(guān)差異表達基因組,從全新的角度探索口腔黏膜白斑惡變的發(fā)病機制,為尋找治療靶點提供依據(jù)。方法:本項研究中采用70umol/L B(a)P和DMBA合劑誘導口腔黏膜白斑細胞DOK,每周加藥一次,間隔加藥4個月,使其惡變?yōu)榭谇击[狀細胞癌細胞OSCC-BD。采用倒置顯微鏡觀察比較兩種細胞的形態(tài)變化,HE染色實驗方法檢測惡變細胞的異型性,Transwell侵襲實驗檢測惡變細胞的體外侵襲能力,碘化丙啶染色及流式細胞術(shù)檢測細胞周期變化,以上實驗方法可用于證實OSCC-BD細胞的惡性特征。采用Affymetrix m RNA基因表達譜芯片篩選口腔黏膜白斑細胞惡變前后的差異基因組,并對差異倍數(shù)較高的基因在兩種細胞系中進行Real-time PCR驗證和分析。結(jié)果:在本次的研究中,我們通過B(a)P/DMBA的合劑成功誘導口腔黏膜白斑DOK細胞惡變?yōu)榭谇击[狀細胞癌細胞OSCC-BD,HE染色結(jié)果顯示與DOK細胞相比,OSCC-BD細胞出現(xiàn)細胞分裂數(shù)增加、接觸抑制喪失、核漿比例增高,以及有散在的多核瘤巨細胞等惡性細胞的特征;通過碘化丙啶染色及流式細胞術(shù)檢測細胞周期變化,與DOK細胞相比,OSCC-BD細胞S期所占比例顯著增加(P0.05),表明OSCC-BD細胞的DNA合成活躍,其增殖和生長能力較DOK細胞顯著增加。通過Transwell實驗顯示OSCC-BD細胞有較強的體外侵襲能力。基因芯片結(jié)果顯示口腔黏膜白斑惡變前后共篩檢出11147個差異基因,其中包含5383個上調(diào)基因和5764個下調(diào)基因,其中非常高倍的差異基因主要與細胞的代謝過程有關(guān)。從差異基因組中挑選出差異倍數(shù)非常顯著的10個基因,在兩種細胞模型中進行初步驗證,結(jié)果提示,有7個基因在細胞模型中表達趨勢與芯片檢測結(jié)果相吻合。結(jié)論:我們通過體外模擬煙草中致癌物B(a)P和DMBA的合劑誘導口腔黏膜白斑細胞惡變,建立了口腔鱗狀細胞癌OSCC-BD的細胞系,并證實了其具有一定的惡性特征。基于這一新的口腔黏膜白斑惡變的細胞模型,進行惡變相關(guān)的差異基因組的檢測和篩選,發(fā)現(xiàn)了一些與腫瘤相關(guān)的新基因,這將為口腔黏膜白斑惡變的預(yù)測和關(guān)鍵治療靶點的探尋提供一個全新的角度和科學線索。
[Abstract]:Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck, accounting for more than 80% of oral malignancies, easy to recur and metastasis, poor prognosis, 5-year survival rate of about 50%, and in recent years there is an upward and younger trend, is a serious threat to human health, more and more people pay attention to it. Oral squamous cell carcinoma (OSCC) often originates from precancerous lesions of oral mucosa, especially oral leukoplakia (OLK), and oral leukoplakia (OLK) is often caused by stimulation of tobacco and other substances. Benzopyrene (a) is the key carcinogen in tobacco. Prene, Bap, and dimethyl-benz [a] anthracene (DMBA), in this study, we first established a new oral squamous cell carcinoma cell model, OSCC-BD cells, which was induced by B (a) P and DMBA in combination with mild to moderate hyperplasia of oral leukoplakia tissue cells (Dysplastic oral keryte, DOK). By studying the cell morphology, cell cycle, growth ability and invasion ability of oral leukoplakia cell line DOK and malignant cell OSCC-BD, the malignant characteristics of OSCC-BD were confirmed. In order to find effective molecular markers for predicting oral leukoplakia malignancy, early detection, early diagnosis, early treatment and improvement of patients with oral leukoplakia malignancy, differential genomics of established oral leukoplakia cells before and after malignancy were studied by gene chip technology. OBJECTIVE: To establish a new cell model of oral leukoplakia malignancy, OSCC-BD cell line, by inducing DOK malignancy in oral leukoplakia cells with B(a) P and DMBA mixture, and to detect the differentially expressed genes associated with malignancy by gene chip technique. METHODS: DOK was induced by 70 umol/L B(a)P and DMBA mixture in oral leukoplakia cells, once a week and at intervals of 4 months. The malignant transformation was observed by inverted microscope. Comparing the morphological changes of the two cells, HE staining test was used to detect the heterogeneity of malignant cells, Transwell invasion test was used to detect the invasiveness of malignant cells in vitro, and propidium iodide staining and flow cytometry were used to detect the cell cycle changes. The above methods can be used to confirm the malignant characteristics of OSCC-BD cells. The differentially expressed genomes of oral leukoplakia cells before and after malignant transformation were screened by expression profiling chip, and the differentially multiplied genes were verified and analyzed by Real-time PCR in two cell lines. OSCC-BD and HE staining showed that OSCC-BD cells showed increased cell division, loss of contact inhibition, increased nuclear-plasma ratio, and scattered multinucleated tumor giant cells compared with DOK cells. The proportion of OSCC-BD cells increased significantly (P 0.05), indicating that the DNA synthesis of OSCC-BD cells was active, and the proliferation and growth ability of OSCC-BD cells were significantly higher than that of DOK cells. Among the 5 764 down-regulated genes, 10 genes with significantly different multiple were selected from the differential genome, and were preliminarily validated in the two cell models. The results showed that the expression trend of 7 genes in the cell model was consistent with the results of microarray detection. CONCLUSION: We established OSCC-BD cell line by simulating the malignant transformation of oral leukoplakia cells induced by a mixture of carcinogens B(a)P and DMBA in tobacco in vitro, and confirmed that OSCC-BD cell line has certain malignant characteristics. The detection and screening of some new tumor-related genes will provide a new perspective and scientific clues for the prediction of oral leukoplakia malignancy and the exploration of key therapeutic targets.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R739.8
本文編號：2212680
[Abstract]:Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck, accounting for more than 80% of oral malignancies, easy to recur and metastasis, poor prognosis, 5-year survival rate of about 50%, and in recent years there is an upward and younger trend, is a serious threat to human health, more and more people pay attention to it. Oral squamous cell carcinoma (OSCC) often originates from precancerous lesions of oral mucosa, especially oral leukoplakia (OLK), and oral leukoplakia (OLK) is often caused by stimulation of tobacco and other substances. Benzopyrene (a) is the key carcinogen in tobacco. Prene, Bap, and dimethyl-benz [a] anthracene (DMBA), in this study, we first established a new oral squamous cell carcinoma cell model, OSCC-BD cells, which was induced by B (a) P and DMBA in combination with mild to moderate hyperplasia of oral leukoplakia tissue cells (Dysplastic oral keryte, DOK). By studying the cell morphology, cell cycle, growth ability and invasion ability of oral leukoplakia cell line DOK and malignant cell OSCC-BD, the malignant characteristics of OSCC-BD were confirmed. In order to find effective molecular markers for predicting oral leukoplakia malignancy, early detection, early diagnosis, early treatment and improvement of patients with oral leukoplakia malignancy, differential genomics of established oral leukoplakia cells before and after malignancy were studied by gene chip technology. OBJECTIVE: To establish a new cell model of oral leukoplakia malignancy, OSCC-BD cell line, by inducing DOK malignancy in oral leukoplakia cells with B(a) P and DMBA mixture, and to detect the differentially expressed genes associated with malignancy by gene chip technique. METHODS: DOK was induced by 70 umol/L B(a)P and DMBA mixture in oral leukoplakia cells, once a week and at intervals of 4 months. The malignant transformation was observed by inverted microscope. Comparing the morphological changes of the two cells, HE staining test was used to detect the heterogeneity of malignant cells, Transwell invasion test was used to detect the invasiveness of malignant cells in vitro, and propidium iodide staining and flow cytometry were used to detect the cell cycle changes. The above methods can be used to confirm the malignant characteristics of OSCC-BD cells. The differentially expressed genomes of oral leukoplakia cells before and after malignant transformation were screened by expression profiling chip, and the differentially multiplied genes were verified and analyzed by Real-time PCR in two cell lines. OSCC-BD and HE staining showed that OSCC-BD cells showed increased cell division, loss of contact inhibition, increased nuclear-plasma ratio, and scattered multinucleated tumor giant cells compared with DOK cells. The proportion of OSCC-BD cells increased significantly (P 0.05), indicating that the DNA synthesis of OSCC-BD cells was active, and the proliferation and growth ability of OSCC-BD cells were significantly higher than that of DOK cells. Among the 5 764 down-regulated genes, 10 genes with significantly different multiple were selected from the differential genome, and were preliminarily validated in the two cell models. The results showed that the expression trend of 7 genes in the cell model was consistent with the results of microarray detection. CONCLUSION: We established OSCC-BD cell line by simulating the malignant transformation of oral leukoplakia cells induced by a mixture of carcinogens B(a)P and DMBA in tobacco in vitro, and confirmed that OSCC-BD cell line has certain malignant characteristics. The detection and screening of some new tumor-related genes will provide a new perspective and scientific clues for the prediction of oral leukoplakia malignancy and the exploration of key therapeutic targets.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R739.8
【參考文獻】
相關(guān)期刊論文 前10條
1 鄭建偉;楊淑娟;利小平;韋從云;李婷;莫文娟;蔡秋云;楊德群;周磊;羅剛;;口腔鱗狀細胞癌及癌旁正常組織基因表達譜芯片檢測[J];中國組織工程研究;2015年27期
2 肖雨;于莉娜;丁彥青;;表達譜基因芯片篩選結(jié)直腸癌轉(zhuǎn)移中Tiam1相關(guān)基因[J];臨床與實驗病理學雜志;2015年04期
3 張琪琪;張志強;溫浩;李秀娟;;利用基因芯片技術(shù)篩選食管癌細胞侵襲轉(zhuǎn)移的相關(guān)分子靶標[J];中國老年學雜志;2015年08期
4 顧文莉;葉冬霞;吳靜靜;;microRNA-125b在口腔鱗狀細胞癌患者血漿中的表達及意義[J];上海口腔醫(yī)學;2015年01期
5 厲周;彭亮;韓帥;黃宗海;史福軍;蔡寨;李秀勤;張普生;朱卉娟;金維榮;;利用Agilent定制基因芯片篩選相同病理類型、不同預(yù)后的早期乳腺癌的分子標記物[J];南方醫(yī)科大學學報;2013年10期
6 燕速;白振忠;趙建信;謝大偉;吳俊麒;;CYP2E1 RsaⅠ基因多態(tài)性與青海地區(qū)人群胃癌易感性研究(英文)[J];青海醫(yī)學院學報;2013年01期
7 顏登國;王國棟;程海玉;;基因芯片技術(shù)分析結(jié)直腸癌肝轉(zhuǎn)移患者免疫基因表達的變化[J];中國普外基礎(chǔ)與臨床雜志;2012年11期
8 劉曉花;李鐵軍;李紓;;口腔黏膜白斑及其惡變相關(guān)因素的研究進展[J];臨床口腔醫(yī)學雜志;2012年07期
9 曾偉;南清振;戴益琛;陳章興;謝軍培;傅育卡;林志翔;姜元芹;朱小三;;利用基因芯片篩查大腸癌腫瘤相關(guān)基因[J];生物醫(yī)學工程與臨床;2010年06期
10 Indranil Chattopadhyay;Sujala Kapur;Joydeep Purkayastha;Rupkumar Phukan;Amal Kataki;Jagadish Mahanta;Sunita Saxena;;Gene expression profile of esophageal cancer in North East India by cDNA microarray analysis[J];World Journal of Gastroenterology;2007年09期
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