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目的:采用蛋白质组学技术筛选结直肠癌(colorectal cancer,CRC)中对化疗药物奥沙利铂(oxaliplatin,L-OHP)耐药的相关蛋白,以期为CRC的个体化治疗提供生物标志物。方法:建立L-OHP耐药细胞株HT-29/L-OHP,提取细胞总蛋白,采用二维凝胶电泳(two-dimensional gel electrophoresis,2-DE)和基质辅助激光解吸电离-飞行时间质谱(matrix assisted laser desorption-ionization time-of-flight tandem masss pectrometry,MALDI-TOF-MS)筛选并鉴定与亲本HT-29细胞差异表达的蛋白,并应用蛋白质印迹法对鉴定获得的部分蛋白进行验证。结果:建立CRC亲本细胞株HT-29和L-OHP耐药细胞株HT-29/L-OHP蛋白的2-DE图谱,共获得表达差异2倍以上的蛋白质点38个,经MALDI-TOF-MS分析,有37个得到鉴定,与亲本细胞相比,表达上调的有17个,表达下调的20个;蛋白质印迹法检测结果显示,多聚胞嘧啶结合蛋白1[poly(C)-binding protein-1,PCBP1]和TUBB2A(tubulin beta-2A)蛋白在HT-29/L-OHP细胞中表达上调,膜联蛋白A3(annexin A3,ANXA3)和磷酸化应激诱导蛋白(stress-induced-phosphoprotein 1,STIP1)表达下调,与MALDI-TOF-MS的实验结果相一致。结论:筛选获得37个与CRC对L-OHP耐药的相关蛋白,为进一步研究CRC对L-OHP耐药机制提供了有力的实验依据。
OBJECTIVE: To screen proteomic related proteins resistant to chemotherapeutic drug oxaliplatin (L-OHP) in colorectal cancer (CRC) by using proteomics techniques in order to provide a biomarker for the individualized treatment of CRC. Methods: The L-OHP resistant cell line HT-29 / L-OHP was established and the total cellular protein was extracted. The two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry The proteins differentially expressed in parental HT-29 cells were screened and identified by matrix assisted laser desorption-ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS). The partial proteins identified were identified by Western blotting. Results: The 2-DE map of HT-29 and L-OHP resistant HT-29 / L-OHP cells was established. 38 protein spots with more than 2-fold difference were established. MS analysis showed that 37 of them were identified. Compared with their parental cells, 17 were up-regulated and 20 were down-regulated. Western blotting showed that poly (C) -binding protein 1, PCBP1] and tubulin beta-2A (TUBB2A) were up-regulated in HT-29 / L-OHP cells. Annexin A3 (ANXA3) and stress-induced-phosphoprotein 1, STIP1), which is consistent with the experimental results of MALDI-TOF-MS. CONCLUSIONS: Thirty-seven related proteins with CRC resistance to L-OHP were screened, which provided a powerful experimental basis for further studying the mechanism of CRC resistance to L-OHP.