Research On The Effect And Mechanism Of 5-Aza-2’- Deoxycytidine Induced Endothelial Progenitor Cells Differentiation

Objective Atherosclerosis cerebral infarction is a major cause of stroke in the elderly, the endothelial cells in atherosclerosis has an important role in the occurrence and development. Studies show that damaged blood vessels of the recovery process is completed by endothelial progenitor cells in pathological condition. Progenitor cells are precursors of endothelial cells, derived from bone marrow of a major precursor cells, which is similar to hemangioblastoma, can proliferate and differentiate into mature endothelial cells. 5-Aza-2’-Deoxycytidine is DNA methyltransferase inhibitors. Epigenetic modifications can drive poorly differentiated precursor cells while preserving portions dry stem cells into endothelial cells. This study was designed to investigate the induction of 5-Aza-d C on EPCs with a view to determine having rapid induction of EPCs into endothelial cells in vitro, and to explore its mechanism.Methods Collected umbilical cord blood, Ficoll-Paque lymphocyte separation medium and 6% hydroxyethyl starch sedimentation mononuclear cells isolated and 10% M199 cultured using immunohistochemistry and inverted phase contrast microscope to identify the cells as endothelial progenitor cells(EPCs), EPCs culture after 7d were added 1μmol / L(d C1 group), medium 3μmol / L(d C2 group) 5-Aza-d C, the negative group intervention for the introduction of 0.0076% DMSO. After 5-Azad C Treatment 0d,3d,7d,14 d, Flow cytometry detect the expression of CD34 +, CD31+.RT-PCR analysis of VE-cadherin, Tie-2 v WF. Western blotting analysis of protein expression of v WF.MSP assay to detect BMP4 gene expression of Cp G island methylation.Results 1. CD34, CD31 and KDR etal by immunofluorescence staining confirmed the EPCs;2. Flow cytometry showed, d C2 group by 5-Aza-d C after 3d 、 7d CD34 +(respectively, 30.2%, 6.7%) were lowercompare with negative group(46.7%, 40.8%) and the difference was statistically significant(?2=5.54,11.17,all P <0.05); d C2 group by 5-Aza-d C after 7d CD31 +(47.9%)were higher compare with negative group(31.3%) and the difference was statistically significant(?2=4.34,P <0.05);3.RT-PCR showed that, d C2 group after treatment 14 d Tie-2, VE- cadherin gene expression were significantly higher than negative group(t =8.12,12.28, all P <0.05); d C1, d C2 group after treatment the 3d, 7d, 14 d Tie-2, v WF, VE-cadherin gene expression are ignificantly higher than 0d(all P <0.05);4.Western blotting analysis showed, d C2 group 0d, 3d, 7d v WF protein were higher than negative group(P <0.05); d C1, d C2 group In the 3d, 7d v WF protein group were higher than the corresponding protein expression in 0 d v WF(all P <0.05);5.After methylated treatment group d C2 and group d C1 BMP4 expression was low, rather than a unmethyl treatment group d C2 and group d C1 BMP4 expression was strong.Conclusion 5-Aza-d C has directed and rapid induction of EPCs into endothelial cells may, this mechanism may promote BMP4 gene expression, and thus stimulate the Tie-2, the role of VE-cadherin and v WF expression.