低剂量氢醌诱导人胚肺成纤维细胞适应性反应的研究

目的　研究氢醌诱导真核细胞适应性反应及其可能的机制。方法　氢醌刺激MRC 5细胞后,用AlamarBlue还原率检测细胞增殖,乳酸脱氢酶漏出率检测细胞死亡,AnnexinV -PI染色检测细胞凋亡。双向凝胶电泳分离细胞总蛋白,图像分析后选取差异蛋白点进行质谱鉴定。结果　AlamarBlue ,乳酸脱氢酶及AnnexinV -PI双染结果显示10 μM氢醌对细胞存活无显著影响,2 5 0 μmol L氢醌显著降低细胞存活率,细胞预先暴露于10 μmol/ L氢醌12h对2 5 .0 μmol/ L氢醌攻击的耐受力明显增强,表现为细胞存活增多,死亡减少。双向凝胶电泳结果显示,对照组MRC 5细胞检测到14. 2 9±36 .9个蛋白点,低剂量组、高剂量组和适应组分别为14 .5 3±30. 7、1191±393和110 .7±2 4 .7。氢醌刺激后2 4个蛋白斑点发生变化,肽指纹图谱及肽序列标签鉴定了其中2 2个蛋白斑点,这些蛋白参与细胞能量代谢、细胞骨架、蛋白折叠、氧化调节、信号传导等各个方面。结论　低剂量氢醌可诱导MRC- 5细胞适应性反应,多种蛋白参与细胞的适应性反应调控。

Adaptive response induced by low concentration of hydroquinone in human embryonic fibroblasts cells

OBJECTIVE: To study the adaptive response induced by hydroquinone(HQ) on eukaryocyte and its possible mechanism. METHODS: After hydroquinone treatment, AlamarBlue reduce rate, LDH release rate were observed to determine the cell proliferation and death. Annexin V/propidium iodide (PI) staining was performed for each treatment to distinguish viable, early, and late apoptosis or dead cells. The total cellular proteins were separated using two- dimensional gel electrophoresis and visualized by sliver staining. Digital images were analyzed using ImageMaster 2D platinum 5.0 software. The differentially expressed protein spots were picked and digested in gel then identified by tandem mass spectrum. RESULTS: The results of AlamarBlue reduce rate, LDH leakage and Annexin V-FITC/PI staining showed that no effect on cell viability was observed at a concentration of 10 micromol/L HQ while 250 micromol/L HQ significantly decreased cell viability. Cells pretreated with 10 micromol/L HQ for 12h show increasing survival to the following expose to 250 micromol/L HQ. In control MRC-5 cells 1429 +/- 369 protein spots were detected and, 1453 +/- 307 in low HQ group, 1191 +/- 393 in high HQ group, and 1107 +/- 247 in adaptive group by two dimensional gel electrophoresis. Twenty-four protein spots showed significant change after HQ stimulation and 22 protein spots were identified by tandems mass spectra. These identified proteins involved in energy metabolism, translation and RNA processing, protein folding, redox regulation, cell structure and cell signaling. CONCLUSION: Adaptive response can be induced by low concentration of HQ on MRC-5 cells and cellular adaptation is a complex process involving in a modulation of diverse cellular functions.