The Study On Simvastatin's Protective Effect Against Inflammatory Of EPCs

Part1:The Culture And Identification Of Endothelial Progenitor Cells From Rabbit Bone MarrowObjective: To study the isolation,culture and amplification in vitro of endothelial progenitor cells from rabbit bone marrow, and observe the process of their growth and differentiation ,then identify their biologic character. In order to establish a complete suit of culture method which is the foundation for further studying the Endothelial Progenitor Cells.Method: Gain mononclearcells (MNCs)from bone marrow by density gradient centrifugation and then separate the EPCs from MNCs with the method of adherence-sieve. Finally proceed the immunofluorescence and immuno-hisrochemistry assay after ten-day culture in the EBM-2 culture medium containing Singlequotes.Results: There can be seen cell colony, formed by the spindle-like adherent cells radiating towards periphery on the fourth day of culture. Cell colony will link with each other, representing the appearance of the cobblestone on the 7～10th day and trabs-like arrangement after tow weeks. Immunofluorescence demonstrates the spindle-like cells have reaction both for DIL-ac-LDL and FITC-UEA-1, with the positive rate of (65±4.0)%, while the rates for the expression of vWF,VEGFR-2,VE-cadh -erin on the spindle-like cells were (90±2.1)%,(77±4.1)%,(78.1±8.2)% respectively.Conclusion: There are rich EPCs existing in marrows. With the method introduced above which is simple and well repeatable we can manage the vitro-culture amplification successfully.Part2:The study on Simvastatin's protective effect against inflammatory injury of EPCsObjective: To investigate the effect of TNF-α(one inflammatory factor) on the E-selectin secretion and eNOS activity of EPCs, and to study the protective effect of different Simvastatin concentration on those changes induced by TNF-α.Method: EPCs cultured in vitro were stimulated with TNF-α(1,10,25,50ng/ml)for 24 hours; the EPCs stimulated by TNF-α(50ng/ml) for 0.5h receive intervention of different Simvastatin concentration(0,0.1,1.0,10μmol/L)lasting 24h. Soluble E-selectin in the supernatant were measured by ELISA and activity of eNOS was measured by chemical colorimetric assay. Results: Under the stimulation of TNF-α, the secretion of E-selectin was increased and the activity of eNOS was decreased (P<0.05). And the change related with TNF-αconcentration. After received the protective of Simvastatin, the expression of E-selectin was decreased and the activity of eNOS was increased compared with control group(P < 0.05).The altered level of E-selectin and eNOS significantly related with Simvastatin concentration. Conclusion: Simvastatin has the protective effect on inflammation damage of EPCs.