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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 compare the changes of related proteins in human lung adenocarcinoma cell line PC-9 before and after the effect of icitidine hydrochloride by proteomic analysis and to explore the mechanism of action of icitin from the protein level. Methods Human EGFR gene mutation detection kit and real-time quantitative PCR instrument were used to detect the EGFR mutation in PC-9 cells. The effect of icitrin on the proliferation of PC-9 cells was detected by MTT assay. Two-dimensional gel electrophoresis (2-DE) The total protein of PC-9 cells and the total protein of PC-9 cells treated with Icitinib were separated to obtain the protein expression profiles. MALDI-TOF-MS Combined with bioinformatics protein identification. Results There was a deletion mutation in exon 19 of EGFR gene in PC-9 cells. In the range of 10-4-10-9 M, ictetin significantly inhibited the growth of PC-9 cells in a concentration-dependent manner. A total of 56 protein spots were detected at the expression level of ≥1.5 times before and after induction. Twenty-nine protein spots with a variance of 1.7 times were selected for MALDI-TOF-MS analysis, and finally 16 proteins were identified. Depending on their function, these proteins can be divided into metabolic enzymes, cytoskeletons, chaperones, signaling molecules, transcription and translation-related proteins. Conclusions 16 proteomes were preliminarily identified by comparative proteomics. These proteins are closely related to the mechanism of action of icitin, and some of them may be new target of molecular therapy.