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目的应用细胞培养稳定同位素标记技术(SILAC)结合生物质谱分析的方法,分析二亚硝基哌嗪(DNP)对6-10B细胞蛋白质表达的影响,并鉴定与转移相关的蛋白质。方法利用SILAC标记6-10B细胞,得到“重标”的6-10B细胞,经DNP处理24 h后提取细胞总蛋白,与未经DNP处理的“轻标”细胞中提取的总蛋白等量混合,酶解肽段,经正交反相色谱分离后用质谱鉴定,对数据进行定性和定量分析。结果共鉴定到2 853个蛋白质,其中172个表达上调,199个表达下调。筛选其中5个与肿瘤转移相关的蛋白质,进行Western blotting验证,证明SILAC定量结果的正确性。通过GO进行分析,从生物过程、细胞定位和分子功能3个方面对差异蛋白进行注释。结论建立了利用SILAC技术研究宿主细胞-化学药物相互作用的方法,发现了DNP处理细胞的关键蛋白质,为探索DNP致鼻咽癌转移的分子机理提供了实验基础。
Objective To investigate the effects of dinitrosopiperazine (DNP) on the protein expression of 6-10B cells by using SILAC combined with bio-mass spectrometry and to identify the proteins involved in metastasis. Methods 6-10B cells were screened by SILAC, and 6-10B cells were screened. The total protein of cells was extracted with DNP for 24 h, and the total protein extracted from the cells without DNP was The protein was mixed in the same amount to digest the peptides and identified by mass spectrometry after being separated by orthogonal reverse phase chromatography. The data were qualitatively and quantitatively analyzed. Results A total of 2 853 proteins were identified, of which 172 were up-regulated and 199 down-regulated. Five of the proteins involved in tumor metastasis were screened and verified by Western blotting to confirm the correctness of SILAC quantification results. Through GO analysis, the differential proteins are annotated from three aspects of biological process, cell location and molecular function. Conclusion The method of using SILAC technique to study the interaction of host cells and chemotherapeutics was established. The key proteins of DNP-treated cells were found, which provided the experimental basis for exploring the molecular mechanism of DNP-induced nasopharyngeal carcinoma metastasis.