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氨甲蝶呤(MTX)是一种重要的常用化疗药物,然而由于肿瘤细胞对其耐药性的增强而经常导致其疗效大大降低。为了寻找并鉴定与MTX耐药性相关的蛋白质从而为进一步阐明MTX的耐药机制提供线索,培养来源于小鼠NIH3T3的小鼠胚胎成纤维细胞系3T3R500与其耐300 mmol/L MTX的细胞系MTX300,提取上述两种细胞系的总蛋白质,双向凝胶电泳分离蛋白质组成分,扫描并通过软件分析考马斯亮蓝染色的2-DE凝胶,选取表达差异最显著的点,胶内酶切后MALDI-TOF-MS进行肽指纹图谱(PMF)鉴定。图像分析显示,实验组和对照组的蛋白质组图谱之间,一些蛋白质点的表达有明显的变化。通过MALDI-TOF-MS和数据库查询,成功鉴定了耐药后表达变化最显著的蛋白质点为二氢叶酸还原酶(DHFR),并通过Western blot验证了该结果,提示DHFR在MTX耐药机制中发挥重要作用。
Methotrexate (MTX) is an important and commonly used chemotherapeutic drug, however, its efficacy is often diminished due to its resistance to tumor cells. In order to find out and identify the proteins related to MTX resistance and provide clues for further elucidating the mechanism of MTX resistance, mouse embryonic fibroblast cell line 3T3R500 derived from mouse NIH3T3 and MTX300 cell line resistant to 300 mmol / L MTX , The total protein of the two cell lines was extracted, the proteome components were separated by two-dimensional gel electrophoresis, and the Coomassie brilliant blue-stained 2-DE gel was scanned and analyzed by software to select the point with the most significant difference. MALDI -TOF-MS for peptide fingerprinting (PMF) identification. Image analysis showed that there was a significant change in the expression of some protein spots between the proteome profiles of the experimental group and the control group. Through MALDI-TOF-MS and database query, the most significant protein spots were identified as DHFR after drug resistance, and the result was confirmed by Western blot, which indicated that DHFR was in MTX resistance mechanism Play an important role.