转化生长因子β1诱导肾小管上皮细胞转分化的蛋白质组学研究

Proteome analysis of rat proximal tubular cells during epithelial to mesenchymal transition induced by TGF-betal

目的研究转化生长因子（TGF）β1诱导肾小管上皮细胞（NRK52E）转分化（EMT）过程中相关靶蛋白的表达变化。方法应用比较蛋白质组学方法。用双向凝胶电泳（2-DE）对TGF-β1刺激组和对照组NRK52E细胞总蛋白进行分离。两组间差异蛋白点用质谱和数据库搜索鉴定，并用RT-PCR和Western印迹在蛋白质和mRNA水平上进一步检测验证。结果鉴定出22个差异蛋白，包括与细胞骨架相关蛋白、参与物质转化和能量代谢的酶类、与蛋白翻译后修饰相关蛋白、细胞因子及其他蛋白等。应用RT-PCR验证了转胶蛋白（transgelin）、ATP合成酶α亚型、苹果酸脱氢酶、丝氨酸（半胱氨酸）蛋白酶抑制因子、泛素结合酶9（Ubc9）和表皮生长因子8在mRNA水平的表达差异，与2-DE结果一致。用Western印迹验证了transgelin、ATP合成酶α亚型两种蛋白的表达差异，亦与2-DE结果一致。结论TGF-β1刺激NRK52E细胞EMT发生过程中可诱导包括与细胞骨架相关蛋白、与翻译后修饰相关蛋白、代谢酶类及细胞因子等多种蛋白表达变化。本研究结果有助于深入理解EMT的具体分子机制及阐明肾脏疾病慢性进展的机制。

Objective To investigate the changes of protein expression in rat kidney proximal tubular epithelial NRK52E cells during the epithelial to mesenchymal transitions （EMT） process. Methods Comparative proteomics was applied. Proteins extracted from NRK52E cells （three plates treated with TGF-β1 and three plates without treatment） were separated by two- dimensional gel electrophoresis （2-DE）. Comparative analysis of 2-DE protein patterns between the two groups were carried out using computerized image analysis. Selected proteins exhibiting significant alternations were identified by mass spectrometry. Western blotting and RT-PCR were performed to examine the expression of the candidated proteins. Results The expression of 22 proteins including transgelin, ARP213 complex 21 000 subunit, destrin, ATP synthase α, ubiquitin- conjugating enzyme 9 （Ubc9）, and malate dehydrogenase involved in the organization of the cytoskeleton, energy metabolism and post-translational modification were altered during the EMT process. Western blotting and RT-PCR confirmed the differences of several altered proteins, which was consistent with the findings of 2-DE. Conclusion TGF-β1 induces specific changes in the expression of structural, regulatory and metabolic proteins relevant to the complex process of EMT in NRK52E cells. This findings are helpful to understand the molecular mechanism of EMT and expound the chronic progress mechanism of the kidney disease.