PPFP基因转染人正常甲状腺细胞Nthy-ori 3-1前后的蛋白质组学研究  

Comparative proteomics analysis of normal thyroid follicular epithelial cell line Nthy-ori 3-1 before and after transfection with PPFP gene

作　　者：刘剑鸣[1] 王志明[2] 李萃[3] 段朝军[3] 邓姣[2] 李新营[2] 

机构地区：[1]湖南省人民医院肝胆外科,湖南长沙410005 [2]中南大学湘雅医院普外科,湖南长沙410008 [3]中南大学湘雅医院卫生部肿瘤蛋白质组学重点实验室,湖南长沙410008

出　　处：《肿瘤》2014年第9期795-802,共8页Tumor

摘　　要：目的 :采用蛋白质组学技术检测转染致瘤基因PPFP前后人正常甲状腺Nthy-ori 3-1细胞中蛋白质组分的改变,筛选PPFP基因调控的蛋白,以期为寻找甲状腺滤泡状癌(follicular thyroid carcinoma,FTC)的标志性蛋白或新的药物靶标蛋白提供线索。方法 :收集Nthy-ori 3-1、Nthy-ori 3-1vector和Nthy-ori 3-1PPFP细胞,提取细胞总蛋白,采用二维凝胶电泳(two-dimensional gel electrophoresis,2-DE)制备3组细胞的总蛋白2-DE图谱;应用PDQuest软件对2-DE图谱进行比较分析,寻找差异表达的蛋白质点;采用基质辅助激光解吸电离-飞行时间质谱(matrix-assisted laser desorption-ionization time-of-l ight tandem mass spectrometry,MALDI-TOF-MS)分析技术对这些差异表达的蛋白质进行鉴定,并选取其中MALDI-TOF-MS鉴定分数较高、覆盖率较大且认为与FTC发生和发展关系较为密切的5种蛋白[prohibitin、半乳糖凝集素1(galectin-1)、细胞角蛋白8(cytokeratin 8)、cytokeratin 19和热休克蛋白27(heat shock protein 27,HSP27)]作为验证对象,采用蛋白质印迹法检测各蛋白在3组细胞中的表达水平。结果 :采用2-DE技术建立了Nthy-ori 3-1、Nthy-ori 3-1vector和Nthy-ori 3-1PPFP 3组细胞的2-DE图谱。应用PDQuest软件分析发现,Nthy-ori 3-1PPFP细胞与亲本Nthy-ori 3-1细胞及空载体Nthy-ori3-1vector细胞中差异在2倍以上的蛋白质斑点有38个,采用MALDI-TOF-MS质谱分析技术结合数据库搜索,共鉴定出了28个差异蛋白质点。Nthy-ori 3-1PPFP细胞与2个对照组相比,表达上调的点有19个,表达下调的点有9个。蛋白质印迹法检测结果与蛋白质组学研究结果完全一致。结论 :PPFP基因转染Nthy-ori 3-1细胞后共筛选获得28个差异表达的蛋白,这些差异表达的蛋白可能是PPFP基因调控的蛋白,为进一步阐明FTC发生和发展的分子机制奠定了基础。Objective： To explore the protein component change in normal thyroid follicular epithelial cell line Nthy-ori 3-1 transfected with oncogene PAX8/PPARgamma fusion （PPFP） gene using proteomics technologies in order to find the proteins regulated by PPFP gene, providing clues to looking for follicular thyroid carcinoma （FTC） marker proteins or proteins as drug targets. Methods： The Nthy-ori 3-1^PPFP cells, Nthy-ori 3-1^vector cells and Nthy-ori 3-1 cells were collected to exract total proteins. The two-dimensional gel electrophoresis （2-DE） maps of total proteins in cells in three groups were created. The 2-DE maps of the three groups were analyzed and compared using PDQuest software to find out differentially expressed protein spots. These differentially expressed candidate proteins were identified by matrix-assisted laser desorption-ionization time-of-flight tandem mass spectrometry （MALDI-TOF-MS）. Five protein spots [prohibitin, galectin-1, cytokeratin 8, cytokeratin 19 and heat shock protein 27 （HSP27）] which were considered to be more closely related to the occurrence and development of FTC and have a higher MALDI-TOF-MS score and a greater coverage were selected and detected by Western blotting. Results： The 2-DE maps of total proteins in Nthy-ori 3-1^PPFP, Nthy-ori 3-1 ^vector and Nthy-ori 3-1 cells were successfully established. Thirty-eight differentially expressed protein spots with a difference in expression more than twice between Nthy-ori 3-1^PPFP cells and the Nthy-ori 3-1 and Nthy-ori 3-1^vector cells were identified by using PDQuest 2-D Analysis Software. Combination of MALDI-TOF- MS and database search identified a total of 28 differentially expressed protein spots. Of all the identified protein spots, 19 proteins were up- regulated and 9 proteins were down-regulated in Nthy-ori 3-1^PPFPceIIs as compared with the Nthy-ori 3-1^vector and Nthy-ori 3-1 cells. This result was fully consistent with the results of proteomics. Conclusion： Twenty-eight differentially expressed pr

关 键 词：甲状腺肿瘤 基因转移技术 蛋白质组学 PPFP基因 

分 类 号：R735.35[医药卫生—肿瘤]