SILAC技术研究DNP对鼻咽癌细胞株6-10B蛋白质组的影响  

SILAC-based proteomic analysis on N,N′-dinitrosopiperazine-mediated nasopharyngeal carcinoma 6-10B cells

作　　者：王巍巍[1] 陈婵[1] 周珊[1] 

机构地区：[1]珠海市人民医院,暨南大学附属珠海医院检验科,广东珠海519000

出　　处：《热带医学杂志》2017年第1期20-25,共6页Journal of Tropical Medicine

摘　　要：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 Study using stable isotope labeling with amino acids in cell culture（SILAC）, coupled with masss pectrometry, was performed on N, N′-dinitrosopiperazine（DNP）-induced 6-10 B cells, Validate the expression levels of some proteins associated metastasis. Methods Labeled with ^2H4-L-lysine and ^13C6^15N4-L-arginine 6-10 B cells were treated with DNP, DNP-induced protein expression was investigated using a quantitative proteomic. Results The SILAC-basedapproach quantified 2 853 proteins, 371 of which showed significant change after DNP treatment（172 up-regulated and 199 down-regulated proteins）. Five proteins of DNP-mediated NPC metastasis were proved by Western blotting, and some signalpathways were analyzed using GO. Conclusion SILAC coupled with LC-MS/MS was used to screen the interaction of the host cell and chemical drug, find key proteins related to DNP- mediated NPC metastasis, This would prove some the molecular mechanism on NPC high metastasis.

关 键 词：鼻咽癌 二亚硝基哌嗪 定量蛋白质组学 

分 类 号：R739.63[医药卫生—肿瘤]