氢醌引起TK6细胞毒性的差异蛋白组学研究

Differential proteomics analysis on hydroquinone-induced cytotoxicity in TK6 cells

目的研究氢醌对人淋巴母细胞株TK6细胞蛋白质表达的影响,探讨氢醌引起细胞应答反应的分子机制。方法采用浓度为20. 0μmol/L的氢醌处理TK6细胞24. 0 h,用蛋白裂解液提取细胞总蛋白并进行定量,以双向凝胶电泳分离蛋白质,图像分析后选取电泳差异点进行胶内酶解,采用基质辅助激光解吸电离串联飞行时间质谱法进行质谱鉴定。以浓度为0. 0、5. 0、10. 0、20. 0μmol/L的氢醌处理TK6细胞24. 0 h,提取总蛋白后,采用免疫印迹法对质谱鉴定出的热休克蛋白70(HSP70)和泛素结合酶2(UBE2N)的蛋白表达进行检测。结果氢醌处理后引起48个蛋白质差异化表达,质谱鉴定出30个表达上调或下调的差异蛋白,功能包括氧化应激、线粒体能量代谢、细胞骨架、细胞周期以及DNA损伤修复等。5. 0、10. 0、20. 0μmol/L氢醌组TK6细胞的HSP70和UBE2N蛋白相对表达水平均高于0. 0μmol/L氢醌组(P <0. 05),与质谱结果一致。结论氢醌能够通过氧化应激引起TK6细胞毒性,由此引发线粒体能量代谢的改变和DNA损伤修复来进行的应答。

Objective To study the effect of hydroquinone on the protein expression on human lymphoblastoid cell TK6,and to explore the molecular mechanism of hydroquinone-induced cellular response. Methods The TK6 cells were treated with 20. 0 μmol/L of hydroquinone for 24. 0 hours. Total protein was extracted by protein lysis buffer and quantified. The proteins were separated by two-dimensional gel electroporthressis. After image analysis,the difference in electrophoresis was selected for enzymatic hydrolysis. The mass spectrometry identification was performed using matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry. The TK6 cells were treated with hydroquinone at a concentration of 0. 0,5. 0,10. 0 and 20. 0 μmol/L for 24. 0 hours,and total protein was extracted. The expression of heat shock protein 70( HSP70) and ubiquitin-binding enzyme 2( UBE2N) of which were identified by mass spectrometry were assayed by western blot. Results A total of 48 differential expression protein spots were detected after hydroquinone treatment,and the mass spectrometry identified 30 differentially expressed proteins with up-or down-regulation. These proteins were related to oxidative stress,mitochondrial energy metabolism,cytoskeleton,cell cycle,DNA damage repair,and so on. The relative expression levels of HSP70 and UBE2 N in TK6 cells of 5. 0,10. 0,20. 0 μmol/L hydroquinone group were higher than those of 0. 0 μmol/L hydroquinone group( P < 0. 05),which was consistent with the mass spectrometry results. Conclusion Hydroquinone can induce cytotoxicity in TK6 cells through oxidative stress,which induces the change of mitochondrial energy metabolism and DNA damage repair.