人肺腺癌细胞系A549线粒体差异蛋白质组学研究

Differential mitochondrial proteomic research on human pulmonary adenocarcinoma cell line A549

目的 研究人肺腺癌细胞系A549与人正常支气管上皮细胞系16HBE线粒体蛋白质组的差异表达.方法 传代培养细胞系A549及16HBE,用线粒体提取试剂盒获取细胞线粒体蛋白质,进行双向凝胶电泳,运用液相色谱串联质谱分析技术筛选出A549和16HBE细胞系线粒体间表达水平显著差异的蛋白,所得结果通过Data Analysis软件标峰,用MASCOT进行结果搜索和数据分析.结果 双向电泳结果显示A549、16HBE细胞系线粒体存在差异的蛋白质点共41个,3倍以上差异的16个,A549细胞系中表达上调的15个,表达下调的26个,其中3倍以上差异表达上调的7个,表达下调的9个.运用液相色谱串联质谱技术鉴定出A549细胞系线粒体表达上调的蛋白质2个：AAA＋ ATP酶家族结构域蛋白3B、tRNA鸟嘌呤糖基转移酶,表达下调的蛋白质7个：热休克蛋白75、复合物Ⅲ亚基1、复合物Ⅲ亚基2、鸟氨酸氨基转移酶、异柠檬酸脱氢酶亚基α、SLP-2、抗增殖蛋白.结论 应用亚细胞蛋白质组学方法,鉴定出肺腺癌细胞系线粒体差异表达蛋白,为阐明肺腺癌发生的分子机制、筛选早期诊断标志物提供了有益的线索.

Objective To investigate the differential mitochondrial protein expressions in human pulmonary adenocarcinoma cell line A549 and normal bronchial epithelial cell line 16HBE.Methods The cell lines A549 and 16HBE were cultured.The cell mitochondria was isolated by mitochondrial fractionation kit.Total proteins of A549 and 16HBE mitochondria were extracted and subjected to twodimensional polyacrylamide gel electrophoresis （2-DE）,and then liquid chromatography-tandem mass spectrometry （LC-MS/MS） was employed to identify the differential proteins.The results was marked peak by data anlasis software,and then the data and analysis results were searched with MASCOT.Results Comparatively analyzing the 2-DE mitochondrial protein patterns of two cell lines,a total of 41 differentially expressed protein spots were found.15 proteins were high-expressed and other 26 proteins were low-expressed in mitochondria of A549.The difference of 16 spots was more than three times,in which seven proteins were high-expressed and nine proteins were low-expressed.Identified by LC-MS/MS,two proteins were high-expressed which were AAA-TOB3 and TGT,and seven proteins were lowexpressed which were HSP75,Complex Ⅲ subunit 1,Complex Ⅲ subunit 2,OAT,ICDHα,SLP-2,and PHB.Conclusions Using the subcellular proteomic method,the differential expression proteins in pulmonary adenocarcinoma cell line are identified.It will provide a uscful clue to elucidate the molecular mechanism of lung cancer and to screen the early diagnostic markers.