新型口腔黏膜白斑恶变细胞模型的建立及恶变相关差异基因组检测的研究

发布时间：2018-08-30 09:34

【摘要】：口腔鳞状细胞癌(Oral squamous cell carcinoma,OSCC)是一种常见的头颈部恶性肿瘤,占口腔恶性肿瘤80%以上,容易复发和转移,预后较差,5年存活率约50%,且近年来有上升和年轻化的趋势,是一种严重威胁人类健康的疾病,越来越引起人们的重视,但该病的发生发展分子机制目前尚不完全清楚。口腔鳞状细胞癌常常起源于口腔黏膜的癌前病变,尤其是口腔黏膜白斑(Oral leukoplakia,OLK),口腔黏膜白斑常因受到烟草等物质的刺激而导致癌变,而烟草中的关键致癌物质是苯并芘(Benzo(a)pyrene,Bap)和二甲基苯并蒽(7,12-dimethyl-benz[a]anthracene,DMBA),在本次研究中,我们首次建立了由B(a)P和DMBA的联合诱导轻中度异常增生的口腔黏膜白斑组织细胞(Dysplastic oral keranticyte,DOK)恶变的新的口腔鳞状细胞癌细胞模型,命名为OSCC-BD细胞;通过对口腔黏膜白斑细胞DOK和其恶变后的细胞OSCC-BD的细胞形态,细胞周期,生长能力和体外侵袭能力等进行研究,从而证实OSCC-BD细胞的恶性特征。尽管早期诊断和治疗对于口腔鳞状细胞癌的预后非常重要,但是其致病机制及相关的有效分子标志物仍旧缺乏,本实验通过基因芯片技术对已建立的口腔黏膜白斑恶变前后的细胞进行差异基因组的研究,以寻找能预测口腔黏膜白斑恶变的有效分子标志物,对口腔黏膜白斑恶变的早期发现、早期诊断、早期治疗及改善患者预后,预防口腔鳞状细胞癌的发生具有重要的科学和临床意义。目的:通过B(a)P和DMBA合剂诱导口腔黏膜白斑细胞DOK恶变,建立新的口腔黏膜白斑恶变的细胞模型即OSCC-BD细胞系,并用基因芯片技术分析检测恶变相关差异表达基因组,从全新的角度探索口腔黏膜白斑恶变的发病机制,为寻找治疗靶点提供依据。方法:本项研究中采用70umol/L B(a)P和DMBA合剂诱导口腔黏膜白斑细胞DOK,每周加药一次,间隔加药4个月,使其恶变为口腔鳞状细胞癌细胞OSCC-BD。采用倒置显微镜观察比较两种细胞的形态变化,HE染色实验方法检测恶变细胞的异型性,Transwell侵袭实验检测恶变细胞的体外侵袭能力,碘化丙啶染色及流式细胞术检测细胞周期变化,以上实验方法可用于证实OSCC-BD细胞的恶性特征。采用Affymetrix m RNA基因表达谱芯片筛选口腔黏膜白斑细胞恶变前后的差异基因组,并对差异倍数较高的基因在两种细胞系中进行Real-time PCR验证和分析。结果:在本次的研究中,我们通过B(a)P/DMBA的合剂成功诱导口腔黏膜白斑DOK细胞恶变为口腔鳞状细胞癌细胞OSCC-BD,HE染色结果显示与DOK细胞相比,OSCC-BD细胞出现细胞分裂数增加、接触抑制丧失、核浆比例增高,以及有散在的多核瘤巨细胞等恶性细胞的特征;通过碘化丙啶染色及流式细胞术检测细胞周期变化,与DOK细胞相比,OSCC-BD细胞S期所占比例显著增加(P0.05),表明OSCC-BD细胞的DNA合成活跃,其增殖和生长能力较DOK细胞显著增加。通过Transwell实验显示OSCC-BD细胞有较强的体外侵袭能力。基因芯片结果显示口腔黏膜白斑恶变前后共筛检出11147个差异基因,其中包含5383个上调基因和5764个下调基因,其中非常高倍的差异基因主要与细胞的代谢过程有关。从差异基因组中挑选出差异倍数非常显著的10个基因,在两种细胞模型中进行初步验证,结果提示,有7个基因在细胞模型中表达趋势与芯片检测结果相吻合。结论:我们通过体外模拟烟草中致癌物B(a)P和DMBA的合剂诱导口腔黏膜白斑细胞恶变,建立了口腔鳞状细胞癌OSCC-BD的细胞系,并证实了其具有一定的恶性特征。基于这一新的口腔黏膜白斑恶变的细胞模型,进行恶变相关的差异基因组的检测和筛选,发现了一些与肿瘤相关的新基因,这将为口腔黏膜白斑恶变的预测和关键治疗靶点的探寻提供一个全新的角度和科学线索。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.8

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