低剂量氢醌、甲醛及过氧化氢刺激人胚肺成纤维细胞适应性反应的共同差异蛋白的表达

目的研究低剂量氢醌(HQ)、甲醛(FA)及过氧化氢(H2O2)刺激引起的细胞耐受与适应反应是否存在共同机制。方法利用已建立的HQ,FA及H2O2诱导人胚肺成纤维细胞MRC-5适应性反应模型,分别选择能诱导产生适应性反应的低剂量和致死剂量的HQ,FA及H2O2交叉染毒,AlamarBlue还原率检测细胞增殖。适应剂量HQ,FA及H2O2处理细胞后,二维凝胶电泳(2-DE)分离细胞总蛋白,图像分析后选取共同变化的差异蛋白点进行质谱鉴定。结果MRC-5细胞交叉染毒结果发现,低剂量HQ预刺激后再给予致死剂量FA或H2O2刺激,以及低剂量FA或H2O2预刺激后再给予致死剂量HQ刺激,其AlamarBlue还原率均较单独用致死剂量HQ,FA或H2O2刺激增高。低剂量HQ,FA和H2O2刺激MRC-5细胞后2-DE图谱图像分析发现,对照组MRC-5细胞检测到1519±154个蛋白点,低剂量HQ组为1551±172个,低剂量FA组为1572±179个,低剂量H2O2组为1429±159个,凝胶匹配后发现,染毒后出现4个共同消失的蛋白点,7个新增蛋白点,经肽质量指纹图谱和串联质谱鉴定了其中6个蛋白点。已鉴定的蛋白包括二磷酸核苷激酶B,蛋白磷酸酶PP1-β催化亚基,Ufm1结合酶1,肌苷-5′-单磷酸脱氢酶,α晶状体球蛋白B链,巯氧还蛋白过氧化物酶1,这些蛋白功能涉及氧化应激、细胞骨架、蛋白折叠等各个方面。结论HQ,FA和H2O2交叉染毒可相互诱导适应性反应;低剂量HQ,FA和H2O2可诱导多种共同蛋白的表达。

Expression of proteins for adaptive response induced by low concentrations of hydroquinone, formaldehyde and hydrogen peroxide in human embryonic lung fibroblasts

AIM To investigate whether the mechanisms of adaptive response induced by low concentrations of hydroquinone (HQ), formaldehyde (FA) and hydrogen peroxide (H2O2) are the same. METHODS According the results of adaptive models established by previous researches, human embryonic lung fibroblasts cells MRC-5, were exposed to adaptive concentration and lethal concentration of HQ, FA and H₂O₂ respectively. Alamar Blue reduce was determined to observe the effect of cell proliferation. The total cellular proteins were separated using two-dimensional gel electrophoresis(2-DE) and visualized by silver staining. Gel images were analyzed using ImageMaster 2D Platinum 5.0. The differentially expressed protein spots were cutted and digested in gel then identified by tandem mass spectrum. RESULTS Alamar Blue reduce showed that cells pretreated with adaptive concentration of HQ, FA or H₂O₂ were more tolerable to following lethal concentration of chemical treatments. In control MRC-5 cells 1519±154 protein spots were detected and, 1551±172 in adaptive concentration of HQ group, 1572±179 in adaptive concentration of FA group, and 1429±159 in adaptive concentration of H₂O₂ group on 2-DE gels. Four spots were disappeared and seven spots were newly appeared after low concentration of ECPs treatment. Six proteins have been identified by peptide mass fingerprinting and/or peptide sequence analysis. Among them are nucleoside diphosphate kinase B，protein phosphatase 1, catalytic subunit, alpha isoform 3, Ufm1-conjugating enzyme 1，inosine-5′-monophosphate dehydrogenase 2， alpha crystallin B chain，peroxiredoxin 1. These proteins are involved in redox regulation, cell signaling, protein folding, and protein modification as well. CONCLUSION Adaptive responses are shown on mutual treatment of HQ, FA and H₂O₂. Low concentrations of HQ, FA and H₂O₂ induce expression changes of some identical proteins in MRC-5 cells.