| objective:Using the method of proteomics to explorer the different expressed proteins of osteoblast,s secreted protein profiling after intervented by icariin in vitro.In order to contributed to lay the foundation for further study of the mechanism of icariin promoting Osteogenesis and development of new drugs.Methods:The ROB were isolated from fetal rats by enzymatie-digestion,and then the passage cells were divided into two groups:the intervention(icariin-treated) group and control group.The end of 24 hours in culture, supernatants were collected from two groups, by ultrafiltration centrifugal- acetone precipitation in the culture medium rich in protein, prepared two-dimensional electrophoresis (2-DE) samples to 2-DE experiments. By 2-DE image analysis software, two groups of cells than the 2-DE protein patterns, looking for differences in protein, and through the matrix-assisted laser desorption ionization- time of flight mass spectrometry (MALDI-TOF-MS) for mass spectrometry analysis, protein peptide mass fingerprinting (PMF), and then use the Mascot software Swiss-Prot database query to identify the type and nature of the protein.Results:Obtained a higher quality of bone cells to secrete the proteins profiles. After Image Master TM 5.0 software comparison found that icariin intervention group than the control group of 60 differentially expressed proteins (11 downregulated,49 Upregulated) expression of the protein are:Max(18kDa protein), Galectin-1,Phosphatidylethanol-amine-binding protein,RhoGDPdissociationinhibitor(GDI)alpha,Peroxiredoxin-6,Hemiferrin,Follistatin-reiated proteinlprecursor+y-actin,Macrophage-cappingprotein,Serpinf 141kDa protein,β-actin, Insulin-likegrowthfactor-bindingprotein2precursor,Alpha-enolase,Prolactin regulatory,element-binding protein,Npat_predicted 38 kDa protein,y-actin,STAM-binding protein,Dlecl 39 kDa protein,Isoforml of Heterogeneous nuclear ribonucleoprotein M, Sfrs2 29kDa protein,Spetex-2E,Biphenyl hydrolase-like,T-complex protein lsubunit theta,Ephrin type-A receptor8,transglutaminase4,Syntaxin-bindingprotein2, Collagen alpha-2(I) chain precursor, Gamma-enolase,Vimentin,ATP synthase subunit beta mitochondrial precursor,protein disulfide isomerase family A, member 3,Argbp2 78 kDa protein,heat shock 60kDa protein 1,Heat shock cognate 71 kDa protein,Lamin-A,inactive dipeptidyl peptidaselO,Collagen alpha-1 (Ⅲ) chain precursor,Filamin-A,Serine-protein kinase ATM,LIM and senescent cell antigen-like-containing domain protein 1. Among them, the 189,194,203 number identified protein was not successful, the function of protein database 169 temporarily not search. Downregulated proteins are:glutaredoxin5, Translationally-controlled tumorprotein, Myosin heavy chain-1, Phosphoglycerate mutase 1,6-phosphogluconolactonase, Cyfipl_predicted 51 kDa protein, Sdha 72kDa protein, CAP-Gly domain-containing linker protein2+Alpha-enolase. Of which 217 identified proteins was not successful. Identified a number of points for the same protein, such as the three points for the Alpha-enolase,3-point position for theγ-actin,2 points for theβ-actin, protein disulfide isomerase family A, member 3 has two spots, suggesting that these proteins may be the existence of different modifications, such as phosphorylation, methylation modified Changed its isoelectric point and molecular weight or due to different monomers. These differences are mainly proteins classified by function cytoskeletal proteins, heat shock protein and molecular chaperone, RNA binding protein and transcription shear extension related protein, signal transduction related proteins, antioxidant enzymes and intermediate metabolic enzymes.Conelusion:By proteomics methods, we found that icariin intervention in rat osteoblasts secreted protein profiling protein expression in a variety of changes, some of which feature is not yet known secreted protein, rich in proteins of rat osteoblasts Group's database for further study of the mechanism of icariin promote bone provides a reliable basis. |
PDF Full Text Request