| Objective: Parathyroid hormone (PTH) is one of the most importanthormones, which regulates calcium homeostasis and bone metabolism.Recently, many investigations have proved that intermittent PTHadministration promotes bone formation, but the mechanism is not clear yet.In order to search for unknown regulation cytokines in bone metabolism, weapplied proteomic methods to investigate the secreted protein profiles of ratosteoblasts (ROB) with or without intermittent PTH treating,. This researchwould not only contribute to the investigation of the molecular mechanism ofPTH effects on osteoblasts but also to the development of new drugs.Methods: The ROB were isolated from fetal rats by enzymatic-digestion,and then the passage cells were divided into two groups: the intermittentlyPTH-treated group (Itm) and the control group (Ctr). Itm groups werecultured in PTH-containing (50ng/ml) medium for the first 6 hours, and in thevehicle medium for the subsequent 18h in a 24h-incubation cycle. The controlgroups were cultured in vehicle medium all the time. There are three cycles inthe whole research. In the third cycle, the cells were cultured in theserum-free medium, and the medium was collected at the end of 6h and 24h.The secreted proteins in the medium were extracted by dialysis-acetoneprecipitation methods, and then they were separated by two-dimensional gelelectrophoresis (2-DE). Subsequently, the differentially secreted proteins inthe two groups of osteoblasts were analyzed by image analysis software.Some of the selected protein spots were digested with trypsin and measuredby matrix assisted laser desorption ionization time-of-flight massspectrometry (MADI-TOF-MS). Finally, the data obtained from peptide massfingerprinting (PMF) was used for protein database search.Results: The reliable 2-DE images were successfully obtained. There are 34 differential protein spots in the intermittently PTH-treated group at 6h(Itm6h) compared to the control group (Ctr6h), while there are 35 differentialprotein spots in the intermittently PTH-treated group at 24h (Itm24h)compared to the control group (Ctr24h). We selected 8 differential proteinspots (4 up-regulated, 4 down-regulated) in Itm6h and 15 differential proteinspots (11 up-regulated, 4 down-regulated)in Itm24h for MADI-TOF-MSanalysis. The results showed that the 4 up-regulated proteins in Itm6h wereUbiquitin specific protease 14, Chloride intracellular channel protein 4,Vimentin, Eukaryotic translation initiation factor 5A-1; the 4 down-regulatedproteins were Neurokinin-B precursor, Alpha-2 antiplasmin (Serine (Orcysteine) peptidase inhibitor, clade F, member 1), Heat shock cognate 71 kDaprotein, Clr protein; and the 11 up-regulated proteins in Itm24h wereVimentin, Glyceraldehyde-3-phosphate dehydrogenase, Rho GDPdissociation inhibitor (GDI) alpha, FK506-binding protein 1A, Pyruvatekinase isozymes M1/M2, Inositol monophosphatase, (IMPase), IGFBP-2,Proteasome subunit alpha type 3, Superoxide dismutase [Cu-Zn]; the 4down-regulated proteins were Glutathione peroxidase 1 (GSHPx-1),Metalloproteinase inhibitor 2 precursor (TIMP-2), Transgelin, Alpha-2antiplasmin (Serine (Or cysteine) peptidase inhibitor, clade F, member 1). Themajority of the modulated proteins belonged to the following functionalcategories: cytoskeleton, regulators of translation, protein degradation,signaling transport, heat shock protein/chaperones, antioxidase and metabolic.Conclusion: We devised a series of methods for the proteome study ofROBs secreted proteins, and obtained reliable 2-DE images. In further study,we found that some of the proteins were first identified as the ROBs secretedproteins. These results may greatly help us to find unknown regulationcytokines in bone metabolism and discover the mechanism of PTH effects. |
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