运用蛋白质组学技术研究与氨甲蝶呤耐药性相关的蛋白质

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闸明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300μmol／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。

Proteomic Analysis for the Identification of Proteins Related to Methotrexate Resistance

Methotrexate(MTX) is one of the most important and frequently used drugs in cancer therapy, but the efficacy of this drug is often compromised by the development of resistance in cancer cells. To seek and identify differentially expressed proteins related to MTX resistance and provide clues for the mechanism of MTX resistance, proteins from cell line MTX300 (resistant to 300 mmol/L MTX) and its control cell line 3T3R500 were separated by two-dimensional electrophoresis (2-DE). The colloidal Coomassie brilliant blue-stained 2-DE gels were subjected to image analysis, which revealed several spots with high levels of dif-ferential expression between MTX300 and 3T3R500. The protein spot with highest differential expression was submitted for tryptic peptide mass fingerprinting(PMF) for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MS analysis and database searches revealed it to be dihydrofolate reductase (DHFR), which was subsequently confirmed by Western blot. The result suggested that DHFR might play an important role in the MTX resistance.