| Objective To investigate the effect of mesenchymal stem cells(MSCs)on the differentiation of endothelial progenitor cells(EPCs)into endothelial cells(ECs).Methods 1.Isolation and culture of murine bone marrow MSCs and EPCs.2.MSCs were plated into the upper and EPCs to the lower chamber of transwell.3.Phase-contrast microscopy was used to examine the changes in cell morphology in the single culture,co-culture,and VEGF groups.4.Immunofluorescence staining,Real-time-PCR(RT-PCR)and Western blotting were utilized to detecte the expression of the endothelial cell markers CD31 and v WF.5.VEGF expression in single culture,co-culture,and VEGF groups was measured via ELISA assay.6.ELISA analysis of VEGF expression in MSC conditioned media(MSCCM).7.EPCs were cultured in MSCCM supplement with anti-VEGF neutralizing antibody;The expression of CD31 and v WF in the LG-DMEM,MSCCM,MSCCM & Ig G,and MSCCM & Anti-VEGF groups were checked.Results 1.EPCs were co-cultured with MSCs for 48 h in vitro.Changes in cell morphology implicated MSCs in the differentiation of EPCs into ECs.Cells in the single culture group were irregularly shaped.By contrast,cells in both the co-culture and VEGF groups displayed a cobblestone appearance.2.Immunofluorescence results showed that EPCs in both the co-culture and VEGF groups highly expressed CD31 and v WF,whereas EPCs in the single culture group weakly expressed CD31 and v WF.RT-PCR analysis showed that the m RNA expression levels of CD31 and v WF were significantly higher in co-culture group(CD31: 5.45 ± 1.45,v WF: 7.50 ± 1.11)than in single culture group(CD31: 1.20 ± 0.10,v WF: 1.15 ± 0.08,**P < 0.01).CD31 and v WF m RNA expression were significantly higher in VEGF group(CD31: 4.29 ± 1.15,v WF: 3.32 ± 0.66)than in single culture group(CD31: 1.20 ± 0.10,v WF: 1.15 ± 0.08,*P < 0.05).Western blot analysis revealed that CD31 and v WF protein expression significantly increased in co-culture group(CD31: 1378.23 ± 67.81,v WF: 141.70 ± 8.50,**P < 0.01)than in single culture group(CD31: 519.87 ± 72.83,v WF: 28.20 ± 3.40,**P < 0.01).CD31 and v WF protein expression levels were significantly higher in VEGF group(CD31: 917.33 ± 19.92,v WF: 69.36 ± 5.45)than in single culture group(CD31: 519.87 ± 72.83,v WF: 28.20 ± 3.40,**P < 0.01).3.VEGF expression in co-culture group is up-regulated.VEGF expression was significantly up-regulated in both the co-culture group(340.94 ± 14.17 pg/ml)and VEGF group(360.67 ± 13.47 pg/ml)than single culture group(85.47 ± 8.80 pg/ml,**P < 0.01).4.MSCs secrete VEGF.VEGF expression was significantly higher in MSCCM(166.03 ± 11.09 pg/ml)than in serum-and factor-free LG-DMEM medium(11.77 ± 3.04 pg/ml,**P < 0.01).5.Immunofluorescence results showed that EPCs in both MSCCM,MSCCM & Ig G groups highly expressed CD31 and v WF,whereas EPCs in the LG-DMEM group weakly expressed CD31 and v WF.And the expression of CD31 and v WF in MSCCM & Anti-VEGF group were lower than MSCCM group.6.RT-PCR analysis showed that the m RNA expression levels of CD31 and v WF were significantly higher in MSCCM group(CD31: 2.85 ± 0.55,v WF: 3.78 ± 0.95)than in LG-DMEM group(CD31: 1.05 ± 0.05,v WF: 1.06 ± 0.07,**P < 0.01).CD31 and v WF m RNA expression were significantly higher in MSCCM group(CD31: 2.85 ± 0.55,v WF: 3.78 ± 0.95)than in MSCCM & Anti-VEGF group(CD31: 1.88 ± 0.16,v WF: 2.30 ± 0.39,*P < 0.05).7.Western blot analysis revealed that CD31 and v WF protein expression significantly increased in MSCCM group(CD31: 278.06 ± 7.52,v WF: 87.13 ± 7.60)than in LG-DMEM group(CD31: 93.80 ± 7.75,29.40 ± 4.32,**P < 0.01).CD31 and v WF protein expression levels were significantly higher in MSCCM group(CD31: 278.06 ± 7.52,v WF: 87.13 ± 7.60)than in MSCCM & Anti-VEGF group(CD31: 176.10 ± 15.41,v WF: 51.76 ± 4.01,**P < 0.01).Conclusion 1.MSCs can promote EPCs differentiation into ECs via co-cultured with EPCs in vitro.2.The mechanism underlying MSCs-mediated EPCs differentiation may depend on paracrine factors such as VEGF. |
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