盐酸埃克替尼对人肺腺癌细胞系PC-9作用的比较蛋白质组学研究  

Comparative proteomics on mechanisms of lung adenocarcinoma cell line PC-9 induced by icotinib

作　　者：刘晓丽[1] 李维[1] 孟夏[1] 张玉萍[1] 明宗娟[1] 史红阳[1] 石婕[1] 钟玉洁[1] 王薇[1] 杨拴盈[1] 

机构地区：[1]西安交通大学第二附属医院呼吸内科,陕西710004

出　　处：《中华肺部疾病杂志（电子版）》2015年第6期10-17,共8页Chinese Journal of Lung Diseases(Electronic Edition)

基　　金：国家自然科学基金资助(81172234)

摘　　要：目的应用比较蛋白质组学方法分析盐酸埃克替尼作用前后人肺腺癌细胞系PC-9细胞相关蛋白的变化,从蛋白质水平探讨埃克替尼的作用机制。方法应用人类EGFR基因突变检测试剂盒和实时定量PCR仪检测PC-9细胞EGFR突变;应用MTT法检测埃克替尼对PC-9细胞增殖的影响;应用双向凝胶电泳(2-DE)技术分别对未经干预的PC-9细胞总蛋白和经埃克替尼作用的PC-9细胞总蛋白进行分离,获得蛋白质表达谱;应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)结合生物信息学进行蛋白质鉴定。结果 PC-9细胞的EGFR基因存在第19外显子缺失突变;在10-4-10-9M浓度范围内,埃克替尼明显抑制PC-9细胞的生长,且呈浓度依赖;埃克替尼诱导前后表达水平≥1.5倍的蛋白点共有56个。选取差异≥1.7倍的29个蛋白点进行MALDI-TOF-MS分析,最终鉴定出16种蛋白质。根据功能,这些蛋白可分为代谢酶类、细胞骨架类、分子伴侣、信号传导分子、转录及翻译相关蛋白。结论应用比较蛋白质组学方法初步鉴定了16种蛋白质,这些蛋白质与埃克替尼作用机制密切相关,部分可能成为新的分子治疗靶点。Objective To analyze icotinib-associated proteins of PC-9 cell with comparative proteomics and bioinformatics technique to discuss molecule mechanism of icotinib. Methods Amoy Dx and real-time PCR were used to detect the EGFR mutation in PC-9 cells gene. MTT was used to investigate theproliferationinhibition oficotinibon PC-9 cells. Two dimensional gel electrophoresis（ 2-DE） was used to separate totally soluble proteins extracted from PC-9 cells either exposed to or not exposed to icotinib,and differentially expressed proteins were analyzed and identified by matrix-assisted laser desorption / ionization time of flight mass spectrometry（ MALDI-TOF-MS）. Proteins were identified by blasting NCBI or SWISS PORT database with Mascot software. Results There was a deletion mutation located in exon 19. In the 10-4-10-9M concentration range,icotiib significantly concentration-dependent inhibited the growth of the PC-9 cells. 56 differentially expressed protein spots（ Ratio ≥1. 5） were found. We finally selected 29 obviously differential protein spots（ Ratio≥1. 7） to analyze by MALDI-TOF-TOF-MS and got 16 proteins successfully. They can be classified into 5 categories based on their functions： enzymes related to cell metabolism,cytoskeleton proteins,signal transduction molecules,chaperone,proteins related to transcription and translation. Conclusions 16icotinib-related proteins are identified by comparative proteomics. These proteins may be associated with antitumor mechanism of icotinib and some of them may become potential drug targets.

关 键 词：PC-9细胞 埃克替尼 比较蛋白质组学 生物信息学 

分 类 号：R563[医药卫生—呼吸系统] R743[医药卫生—内科学]