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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 detect the changes of protein components in normal thyroid Nthy-ori 3-1 cells before and after transfection with tumorigenic PPFP by proteomics, and to screen the protein regulated by PPFP gene in order to find out the potential of follicular thyroid carcinoma, FTC) or new drug target proteins. Methods: Nthy-ori 3-1, Nthy-ori 3-1vector and Nthy-ori 3-1PPFP cells were collected and total cellular protein was extracted. Three groups were prepared by two-dimensional gel electrophoresis (2-DE) The total protein 2-DE map of the cells was analyzed. The 2-DE maps were analyzed by PDQuest software to find differentially expressed protein spots. The matrix-assisted laser desorption-ionization time-of- These proteins were identified by MALDI-TOF-MS and analyzed by MALDI-TOF-MS. The MALDI-TOF-MS was used to identify the proteins with high MALDI-TOF-MS identification score and high coverage and was found to be closely related to the occurrence and development of FTC Of prohibitin, galectin-1, cytokeratin 8, cytokeratin 19 and heat shock protein 27 (HSP27) were used as validation subjects, and Western blotting Method to detect the expression of each protein in three groups of cells. Results: The 2-DE pattern of Nthy-ori 3-1, Nthy-ori 3-1 vector and Nthy-ori 3-1 PPFP 3 cells was established by 2-DE technique. The results of PDQuest software analysis showed that there were 38 protein spots with more than 2-fold difference between Nthy-ori 3-1PPFP cells and Nthy-ori 3-1 cells and empty vector Nthy-ori3-1vector cells. MALDI-TOF-MS A total of 28 differential protein spots were identified by mass spectrometry combined with database search. Nthy-ori 3-1PPFP cells had 19 up-regulated spots and 9 down-regulated spots compared with the two control groups. The result of Western blotting is completely consistent with the result of proteomics research. CONCLUSIONS: Twenty-eight differentially expressed proteins were co-screened by PPFP gene transfection in Nthy-ori 3-1 cells. These differentially expressed proteins may be regulated by PPFP gene, which lays the foundation for elucidating the molecular mechanism of the occurrence and development of FTC .