| Objective:To understand the effect of high glucose on rat glomerular mesangial cells (MCs) protein through observing their dynamic proteomics change under high glucose environment.Methods:1. Primary culture and identification of mesangial cells: the mesangial cells were cultured from rats by demixing filtration,and the cellular purity identification was carried out by immunocytochemistry with anti-Thy1.1, anti-α-SMA,Cytokeratin,vWF antibodies respectively.2. The 5th passages mesangial cells after primarily culture were cultured in vitro in different mediums including normal glucose level (NC group, contain glucose 5.5mM) and high glucose level(HG group,contain glucose 30 mM). The total proteins were extracted after cells gathered at 0 hour,8 hours,16 hours,72 hours,25days. They were segregated using the technique of two-dimensional gelelectrophoresis. Then the gels were stained by colloid coomasbrilliant blue in order to reveal the segregation result.3. 2-DE images were analyzed digitally by utilizing ImageMaster 2D Platinum 6.0 analysis software. The statistical method was applied to assess reliability and reproducibility of the 2DE result. If p value from heterogeneity of variance t-test is less than 0.05 after FDR calibrationand volume% of protein spots is greater than 2-fold change ,it was regarded as differential protein spots.4. The differential protein spots in the gels were cut,then the peptide mass fingerprint were obtained through In-gel trypsin digestion and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF- MS) analysis.5. The acquired MS data was searched using the Mascot database after trypsin autodigestion and keratin peaks were removed.We judge the protein spots according to the criterion that scores is greater than 60, sequence coverage is greater than 20%,molecular weight (MW) discrepancy is less than±30% and isoelectric point (PI) discrepancy is less than±2.0 between the calculated value and the measured value.6. The proteins identified were classified by GoMiner software based on the Gene Ontology(GO) database. The result revealed through searching the function of these proteins in PUBMED database.Results:1. The primary glomerular Mesangial cells (MCs) were cultured successfully from rats.The immunocytochemistry result of cultured cells showed that MC was positive for specific expressed antigen Thy 1.1andα-SMA,and positive rate is 100%,and for Cytokeratin and vWF expressed positively in the epithelial cells and endothelial cells respectively, MC was negative and negative rate is 100%.2. The good 2-DE results were achieved in reproducibility and reliability.Taking the NC group at 16 hours as study subject,average spots of 3 gels were 965.33±11.37 and 810 spots were all revealed in 3 gels. The matched rate was 84%. The result indicated that the average coefficient of variation (CV) for volume% in 3 gels was 17%-18%.3. There are 27 non-redundant differential protein spots between NC and HG group at different time. In HG group,2 increased and 23 decreased in obvious manners at early stage (8 hours and 16 hours), besides 2 decreased significantly,the rest have no statistically significant compared with NC group at middle stage (72 hours), However, there are 2 spots that constantly stable expressed at previous time increased highly under the effect of high glucose for a long time (25 days).4. Mass charge ratio and intensity information of these 27 differential protein spots were obtained entirely.Among which 26 spots consistented with above mentioned criterion through searching in MASCOT database. The 12th and 23th spots were mixture of two kind of proteins. The 5th and 24th spots, the 15th and 16th spots were the same protein respectively. They may caused by deviation of different PI and MW due to different modification.The correct judgement probability of the 18th spot was higher even if it does not completely conistented with above mentioned criterion.5. Based on GO classification,bioinformatics analysis was carried out for the 22 proteins except the 14th spot without annotated gene and the 12th,23th spots in view of their mixed attribute.The differential proteins of MC under high glucose condition mostly located in cytoplasm, nucleus,cytoskeleton,mitochondria and endoplasmic reticulum. Their molecular function were mainly binding with protein,nucleic acid,ion and have the activity of antioxidant,oxidation-reduction,hydrolytic enzyme,isomerase. They primarily involved in the biological process such as cell communication,cell cycle,apoptosis,proliferation,and metabolic process of carbohydrate,DNA,RNA,protein,oxygen and active oxygen etc.6. By means of consulting literature,It was proposed that decreased proteasomes degradation function may play an important part in the process of diabetic nephropathy.Conclusion:The differential proteomics analysis initially indicated that MC proteome dynamic express information caused by high glucose. Subcellular localization,molecular function and main related biological process of these differential expressed proteins were obtained through bioinformatics analysis. It was proposed that decreased proteasomes degradation function may play an important part in the process of diabetic nephropathy.
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