Emps The Initiative Role In Study Of Huvecs And Seldi Screening Differences In Protein Secondary Appraisal

Objectives:To study the effects of different concentrations of endothelial microparticles (EMPs), which were in response to cellular activation and apoptosis, on proliferation, apoptosis, migration, permeability of endothelial cells and angiogenesis.Methods:Human umbilical vein endothelial cells (HUVECs) were used to generate EMP, acEMPs were in response to TNFa-induced activation and apEMPs were in response to A23187-induced apoptosis. EMPs counts and phenotype of CD62E assay were determined by flow cytometry. BrdU cell proliferation assay, annexin V-FITC apoptosis detection kit, the "scratch assay", transwell system and capillary-like tube formation assay were used respectively to measure the proliferation, apoptosis, migration, permeability and angiogenesis ability of the HUVECs after incubating with105/mL,104/mL and103/mL of acEMPs and apEMPs,respectively.Results:1. There were significant differences between acEMPs and apEMPs in phenotypical and quantity analysis. It was higher in activation group of both EMPs counts and CD62E-positive rate (p<0.05).2. The acEMPs largely promoted the proliferation of HUVECs at high concentrations but apEMPs slightly inhibited it, and both of the effects were concentration-dependent.3. Neither acEMPs nor apEMPs had significant effect on apoptosis of the HUVECs.4. Only apEMPs could enhance the migration of HUVECs at the concentration of104/mL.5. The apEMPs highly elevated the permeability of HUVECs in a concentration-dependent way, and the effect is maximized at the concentration of104/mL; while acEMP suppressed the permeability of HUVECs at a higher concentration but elevated it at lower concentrations.6. The acEMPs could largely inhibit the angiogenesis of HUVECs; but apEMPs could slightly inhibit that only at low concentrations.Conclusions:There are significant differences between the effects of acEMP and apEMP on proliferation, apoptosis, migration, permeability of endothelial cells and angiogenesis, which give us useful information to get a better understanding of effects of EMPs on impairment, repair and related functions of endothelial cells. Objectives:To further separate and identify the differentially expressed proteins analyzed by SELDI, and discuss the methodology significance.Methods:Endothelial progenitor cells cultured in the medium adding with melatonin at concentration of10μM were used to extract proteins, which were analyzed by SELDI for preliminary screening differentially expressed proteins. Tricine-SDS-PAGE and2-DE were chosen to separate the target proteins which had been screened out by the SELDI, respectively, and finally Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) was used to identify the proteins.Results:A protein with molecular weight of53kDa had been separated by Tricine-SDS-PAGE, which was futher identified as a-tubulin by FTICR-MS (score:87); and a high expressed protein with molecular weight of72～95kDa near acid side of the gel had been separated by2-DE, which was finally identified as human heat shock protein90-α by FTICR-MS (score:91)Conclusions:Tricine-SDS-PAGE is preferred when separate proteins in the mass range of35-70kDa with good repeatability and low expenditure of samples, while2-DE is better for separating bigger proteins, and it can provide informations of Molecular Weight and Isoelectric Point but have strict demand for sample loading. Electrospray ionization (ESI) combined with FTICR-MS provides high resolution and stable results.