双向凝胶电泳-飞行时间质谱法分析促癌剂TPA诱导NIH3T3细胞表达变化的蛋白质

Analysis of Proteins with Altered Expression Induced by Tumor Promoter TPA in NIH3T3 Cells by Two-Dimensional Electrophoresis and MALDI-TOF-Mass Spectrometry

[目的]寻找并鉴定在TPA处理后小鼠成纤维细胞NIH3T3中表达发生变化的蛋白质,为阐明TPA在诱导细胞发生生物学变化的作用机制提供线索。[方法]用TPA刺激NIH3T3细胞,分别提取实验组和对照组的NIH3T3细胞的总蛋白质,通过聚丙烯酰胺凝胶双向电泳,硝酸银染色后用软件分析寻找差异表达的蛋白质,并对差异蛋白质进行质谱鉴定。[结果]软件分析显示TPA处理后的NIH3T3细胞中有明显的差异表达蛋白质,质谱鉴定表明TPA处理后,表达上调的蛋白质有线粒体基质蛋白P1前体、微管蛋白beta-5链;TPA处理后明显表达下调的蛋白质有galectin-1、N-myc下游调节基因1蛋白。[结论]TPA刺激NIH3T3细胞后可以下调生长抑制基因的表达,同时上调与细胞增生相关基因的表达。因此,促癌剂TPA可能对NIH3T3细胞有促进增生的作用。

[Objective] Phorbol ester-TPA, which is closely associated with tumorigenesis as a promoter, can induce complicate biological changes in cells by unknown mechanism. The aim of this study was to find out and identify the proteins with altered expression in mouse fibroblast-NIH3T3 cells after TPA treatment, and provide clues to clarify the molecular mechanism of biological changes induced by TPA. [Method] The total proteins were extracted from NIH3T3 cells of experiment group and control group, and the proteins with altered expression were detected by analysis with software following 2-D PAGE and silver staining. Then those different proteins were identified by mass spectrome-try. [Result] There were obviously some proteins with altered expression in NIH3T3 cells after TPA treatment. Identification by mass spectrometry showed that TPA can upregulate the expression of proteins-' mitochondrial matrix protein p1 precursor' and tubulin beta-5 chain', and downregulate the expression of proteins-'galectin - 1' and 'NDRG1'. [Conclusion] TPA can induce NIH3T3 cells to downregulate the expression of growth inhibition genes, and upregulate the expression of some genes associated with proliferation. The treatment with tumor promoter TPA can contribute to proliferation of NIH3T3 cells.