论文部分内容阅读
为筛选鼻咽癌的甲基化沉默基因,采用二维凝胶电泳(2-DE)技术分离甲基转移酶抑制剂5-杂氮-2′-脱氧胞苷(5-aza-2-dC)处理与未处理鼻咽癌细胞5-8F的蛋白质,PDquest图像分析软件识别差异蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异蛋白质.然后采用Westernblotting和RT-PCR检测差异蛋白质nm23-H1在药物处理与未处理5-8F细胞中的表达水平,采用甲基化特异性PCR(MS-PCR)检测nm23-H1基因在药物处理与未处理5-8F细胞中的甲基化水平.建立了5-aza-2-dC处理与未处理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处理后的5-8F细胞中表达上调,而18个蛋白质表达下调.Westernblotting和RT-PCR结果显示,nm23-H1在5-aza-2-dC处理5-8F细胞后表达上调,MS-PCR结果显示,在5-aza-2-dC处理5-8F细胞后nm23-H1基因甲基化水平下降,结果证实,nm23-H1基因是5-8F细胞中的甲基化沉默基因.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,为筛选鼻咽癌甲基化失活基因提供了科学依据.
To screen methylation-silencing genes of nasopharyngeal carcinoma, the methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-2-dC ) Were used to treat 5-8F protein in nasopharyngeal carcinoma cells, PDquest image analysis software was used to identify differential protein spots, and matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to identify differentially expressed proteins.Western blotting and RT- To detect the expression of nm23-H1 protein in drug-treated and untreated 5-8F cells, methylation-specific PCR (MS-PCR) was used to detect the expression of nm23-H1 in drug-treated and untreated 5-8F cells Methylation level.The 2-DE map of 5-aza-2-dC-treated and untreated 5-8F cell proteins was established, 49 differentially expressed protein spots were identified, and 33 differentially expressed proteins were identified, including Fifteen proteins, including nm23-H1, were upregulated in 5-8F cells treated with 5-aza-2-dC and 18 were down-regulated.The results of Western blotting and RT-PCR showed that nm23- The expression of nm23-H1 gene in 5-8F cells treated with 5-aza-2-dC was up-regulated by 2- The results showed that the nm23-H1 gene is a methylation-silencing gene in 5-8F cells.The gene upregulated after 15 5-aza-2-dC treatment may be methylation silencing in 5-8F cells Gene, for screening nasopharyngeal carcinoma methylation inactivation gene provides a scientific basis.