【作者】 张文静；

【导师】 肖志强；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 鼻咽癌(NPC)是我国南方地区常见的一种恶性肿瘤,其发病机制还不清楚。DNA甲基化修饰是基因表达调控的重要表观遗传学机制之一,DNA甲基化修饰导致抑瘤基因等的沉默在癌变过程中发挥重要的作用。因此,筛选鼻咽癌的甲基化失活基因将有助于揭示鼻咽癌的发病机制,具有重要的理论和应用价值。本研究以高转移的鼻咽癌细胞系5-8F为对象,采用蛋白质组学技术筛选鼻咽癌细胞的甲基化失活基因。首先用MTT比色法和流式细胞仪分析确定去甲基化药物5-杂氮-2’-脱氧胞苷(5-aza-2-dC)处理5-8F细胞的最适浓度。然后应用双向凝胶电泳(2-DE)技术分离5-aza-2-dC处理与未处理5-8F细胞的蛋白质;利用PDQuest图像分析软件比较两者2-DE图谱的异同,识别差异表达的蛋白质点;应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异表达的蛋白质;采用Western blot和RT-PCR检测差异蛋白质nm23-H1的表达水平;再采用甲基化特异性PCR(MSP)检测5-8F细胞中nm23-H1基因的甲基化。主要结果如下:1.采用蛋白质组学技术建立了5-aza-2-dC处理与未处理鼻咽癌细胞株5-8F细胞蛋白质的2-DE图谱,识别了49个差异表达的蛋白质点,鉴定了33个差异表达的蛋白质,其中包括nm23-H1在内的15个蛋白质在5-aza-2-dC处理后表达上调,而18个蛋白质表达下调。15个5-aza-2-dC处理后表达上调的基因可能是5-8F细胞中的甲基化沉默基因。2.Western blot结果显示:在5-aza-2-dC处理5-8F细胞后,nm23-H1蛋白质的表达上调,这与蛋白质组学的研究结果一致。3.MSP结果显示,5-aza-2-dC处理5-8F细胞后,nm23-H1基因启动子甲基化水平降低,而非甲基化水平增加。RT-PCR结果显示,5-aza-2-dC处理后,nm23-H1基因mRNA的表达明显上调。结果说明,nm23-H1基因是5-8F细胞中的甲基化沉默基因。本研究首次采用蛋白质组学技术,从蛋白质组水平筛选鼻咽癌细胞的甲基化失活基因,鉴定了15个候选的鼻咽癌细胞的甲基化失活基因,证实鼻咽癌细胞的nm23-H1基因存在甲基化沉默,可能在鼻咽癌发病中具有重要作用。本文结果为研究鼻咽癌的甲基化失活基因提供了实验依据,为筛选与肿瘤发生发展相关的甲基化失活基因提供了新思路。更多还原

【Abstract】 Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China. However, the mechanism underlying the pathogenesis of NPC remains unclear. DNA methylation is one of the epigenetics mechanisms of gene expression regulation. And promoter methylation of tumor suppression genes plays a critical role in carcinogenesis. Therefore, screening methylation silenced genes may help to reveal the pathogenesis of NPC, which has important theoretical and clinical value.Proteomic analysis was performed to screen for methylation silenced genes in NPC cell line 5-8F with high metastatic potential. MTT assay and flow cytometry were used to determine the optimum concentration of demethylating agent 5-aza-2-dC, which was used to treat 5-8F cells. Two-dimensional gel electrophoresis (2-DE) was performed to separate the proteins of treated and untreated 5-8F cells with 5-aza-2-dC. PDQuest software was used to analyze 2-DE images to determine the differential expression proteins between the treated and untreated 5-8F cells. MALDI-TOF-MS was used to identify the differentially expressed proteins. Western blot and RT-PCR analysis were used to examine the expression levels of the differential expression protein nm23-H1. Methylation-specific PCR (MSP) was performed to examine the methylation status of nm23-H1 gene in 5-8F cells. The results were as following: (1) 2-DE reference patterns of 5-8F cells treated and untreated with 5-aza-2-dC were established. Forty-nine differential protein spots were found between treated and untreated 5-8F cells, and thirty-three non-redundant proteins were identified. Among them, 15 proteins including nm23-H1 were up-regulated, and 18 proteins were down-regulated after treated with 5-aza-2-dC. Encoding genes of 15 up-regulated proteins may be methylation silenced genes in 5-8F cells; (2) Western blot analysis showed that the expression of nm23-H1 was up-regulated in 5-8F treated with 5-aza-2-dC, which was consistent with the result of proteomic analysis; (3) MSP showed that methylation level of nm23-H1 promoter was decreased, while unmethylation level of that was increased after treated with 5-aza-2-dC, and RT-PCR showed that nm23-H1 mRNA expression was increased obviously after treated with 5-aza-2-dC, which suggested that nm23-H1 is methylation silenced gene in 5-8F.In this study, it is the first time using proteomic analysis to screen for methylation silenced gene in NPC. 15 candidate methylation silenced genes in NPC cells were identified, and our data suggested that nm23-H1 is a methylation silenced gene in NPC cells, which may play a critical role in the pathogenesis of NPC. The results will be helpful for further investigating methylation silenced genes in NPC, and provide a new idea for screening methylation silenced genes involved in the pathogenesis of tumor.更多还原