Proteomic Differential Display Analysis Of Diabetes Serum And High Glucose Load U-937Cell Line

Objectives:To analyze diabetes patients’serum and the affection of key protein expression by vitamin D3, in order to investigate the interaction mechanism of changed proteins and diabetes. And to investigate the molecular mechanisms of VD applied to diabetic population, and to provide a scientific basis for controlling diabetes.Methods:(1)10fasting blood serum samples of type2diabetics were selected as study group and another10samples from health examination people as control group. The serum samples were extracted and separated by2-DE, differential expression proteins were identified by MALDI-TOF-MS.(2) Good status U-937cells were cultured by high glucose medium which contained45mmol/L D-Glucose, and treated by1,25-(OH)2-D3whose final density was1.6×10-4mmol/L. The high-glucose control group were U-937cells cultured by high glucose. The normal control group was U-937cells cultured normally. The whole cell proteins were extracted and separated by2-dimentional gel electrophoresis (2-DE), differential expression proteins were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS).Results:(1) There were6protein spots whose expression was significantly different between type2diabetes group and the control group. Four protein spots were identified by MALDI-TOF-MS. Those proteins are clusterin precursor, CRP precursor, prohibitin, and catalase.(2) During U-937cells experiments there were thirty protein spots whose expression was significantly different between vitamin D-treated and untreated high glucose loaded cells. Thirteen protein spots were identified by MALDI-TOF-MS. Those proteins are prohibitin, TCP, cargo selection protein TIP47, catalase, protein disulfide isomerase, purine nucleoside phosphorylase, stathmin, electron transport flavoprotein subunit alpha, fumarylacetoacetate hydrolase,71Kd heat shock cognate protein, actin cytoplasmicl, triosephosphate isomerase, and mitochondrial ATP synthase subunit D.Conclusion:Two-dimensional gel electrophoresis and MALDI-TOF-MS were used to identify the differentally expressed proteins between diabetes and the control population, and between high glucose load U-937cell stimulated by Vitamin D and the control groups.These differential proteins are mainly related to oxidative stress and energy metabolism. These data proved that high glucose environment in diabetes is closely related to inflammatory response. These data also suggest that1,25-(OH)2-D3might regulate cell high glucose load by reduction oxidative stress injury and affection energy metabolism.