| Hydroquinone(HQ) is the major metabolite of benzene and plays an important role in benzene-induced toxicity. It also serves as a frequently used chemical material and an important solvent in industry. HQ is absorbed from skin, alimentary canal and respiratory system, and mainly metabolized in the liver. It is expelled into urine conjugated with glucuronate or sulfate, but it can be partly accumulated in human bodies and damage liver, kidney, hematopoietic system, immunity system, central system and so on. Therefore, HQ's toxity on human especially on occupational workers has aroused wide concern.Many studied have been carried out to explore the mechanism of HQ toxicity in cell level, protein level and gene level. It was reported that HQ could induce cell apoptosis, depress cell proliferation, induce chromosome aberration and DNA damage, induce the protein expression changes, and so on. However, most of those studies were confined to one or a few targets, and could not give a fully description of the mechanism. Recently high-throughput screening techniques have developed quickly, which provide new ideas for mechanism study and are widely applied in preventive medicine study as a useful method in screening specific protein marker. Therefore, in this study, we used 2-DE and mass spectrum method to investigate the mechanism of HQ toxicity on bone marrow mesenchymal stem cells. We expect to identify some responsive proteins and dissect the mechanism of HQ toxicity, thus provide effective molecular markers for disease prevention or cure.Bone marrow was collected in the First Hospital attached to Zhejiang University. The mesenchymal stem cells were then isolated through density gradient centrifugation and then subcultured. The third generation cells were exposed to HQ for 24 h at 5×10-5 mol/L concentration, while the control group was treated with the same concentration phosphate buffered saline. Immediately after the treatment, total cellular proteins were extracted and the concentrations were determined with Bradford assay. Proteins from all samples were separated using 2-dimentional gel electrophoresis (2-DE) with pH gradient range of 5-8 in the first dimension and 12.5% SDS-PAGE gel in the second dimension (Bio-Rad). Proteins were detected by silver staining and spot distribution pattern was analyzed using PDQuest 8.0 software package. The experiments were totally repeated for 3 times with 3 repetitions for each group. The results showed that 80% spots are matched between the exposure group and the control group. Among three replications, there are 11 differential spots, in which 4 spots are up regulated while 7 spots are down regulated. Five well-repeated differentially expressed protein spots were cut from the gels and subjected to in-gel digestion with trypsin. The digested peptides were analyzed with a QSTAR Elite LQ-Qq-TOF coupled with a HPLC system. Mass spectrometry data were applied to the program proteinpilot 2.0. Through searching the NCBI human protein database, five proteins were identified which were Annexin-A2, UDP-glucose dehydrogenase, UDP-glucose 6-dehydrogenase, aldolase A and Esterase D (formylglutathione hydrolase).The expressions of Annexin-A2 in the control group and the HQ-treated group were detected using Western Blot analysis. Data showed that the expression level of Annexin-A2 was greatly reduced after HQ treatment, which corroborated that HQ could reduce Annexin-A2 expression.Based on the data, the conclusions drawn from the thesis are:1. HQ induces the protein expression profile changes of bone marrow stem cells at 5×10-5 mol/L concentration. 11 differential spots were identified after HQ treatment, in which 7 spots were down-regulated and 4 spots were up-regulated.2. Annexin-A2, UDP-glucose dehydrogenase, UDP-glucose 6-dehydrogenase and aldolase A, were identified as down-regulated proteins in this study, while Esterase D were identified as up-regulated proteins.3. HQ-induced down-regulation of Annexin-A2 expression was confirmed using Western Blot method. |
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