Sdf-1a In Rat Bone Marrow-derived Cd133 ~ + / - Of Vegfr-2 ~ + / Cd34 ~ + Epcs, Proliferation, Migration And Other Activity

Aim: To explore a new way to purify EPCs, Hybridoma dish was used to isolate EPCs from rat bone marrow -derived cells,according to the morphology of endothelial progenitor cells colony-forming units (EPCs-CFUs) and special markers of EPCs.Methods: The bone marrow derived cells, from rat femoral bone and shinbone, were plated and cultured on Hybridoma dish which was made of polystyrene. Between 4th and 7th days in culture, Stem cells colony-forming units were picked out respectively under microscope. Then one part of this cells was identified by immunofluorescence staining of CD133+VEGFR-2+ , the special markers of EPCs. If the both markers of this part was positive,the rest part of the cells was continuely cultured for passaging.We named this method as"Micropore-Method".Results: The conspicuous stem cells colony-forming units were observed under microscope after four days in cluture, about 7% CFUs were CD133+/VEGFR-2+ positive EPCs-CFUs. After further culturing 7 days, Purity of cells with special markers of CD133+/VEGFR-2+/CD34+ was over 70% identified by flow cytometry. Passaging cells can form capillary tube like formation and differentiate to endothelial like cells expressing EC special marker vWF.Conclusion: It can be concluded that"Micropore-Method"is a new successful way to isolate EPCs from rat bone marrow. Aim:To study whether stromal cell-derived factor-1α(SDF-1α) have effects on endothelial progenitor cells (EPCs) and its preliminary mechanism.Methods : Bone marrow derived CD133+/VEGFR-2+/CD34+EPCs were aquried by Micropore-Method and characterized by immunofluorescence staining and Flow cytometry. The abilities of proliferation, migration and adherence, colony-forming units and capillary tube like formation were detected by the methods of MTT, transwell migration, adherence assays and cloning experiment after EPCs treated with different concentrations of SDF-1α. Cell apoptosis were detected by the methods of Hoechst 33258 staining and flow cytometry after EPCs apoptosis induced by Ox-LDL. CXCR4 mRNA and protein level were determined by reverse trancriptase -polymerase chain reaction (RT-PCR) and Western blot.Results:SDF-1αpromoted the biologic activity of proliferation, migration ,adherence, colony-forming units and capillary tube like formation in a concentration-dependent manner (n=3,P<0.05 or P<0.01 ), SDF-1αconspicuously decreased cell apoptosis after treated with Ox-LDL, SDF-1αreduced apoptosis of EPCs from 22.1±1.80% to 13.37±1.464% (n=3, P<0.01). The expression of CXCR4 mRNA and protein increased in a concentration-dependent manner.Conclusion: SDF-1αimproved the biologic activity of EPCs and reduced EPCs apoptosis which induced by Ox-LDL,it correlated with upregulation expression of CXCR4.