Experimental study on cultured bone marrow endothelial progenitor cells transfected with gene of human hypoxia inducible factor-1? in vitro / 吉林大学学报(医学版)

ABSTRACT

Objective To transfect PCDNA3.0-HIF1-? into the endothelial progenitor cells(EPCs),in order to provide the foundation for exploring myocardial vascular repairing capacity and angiogenesis of the EPCs transfected with HIF gene.Methods The EPCs were separated by the method of density gradient separation in the human bone marrow,and then the cells were inoculated in the fibronectin-packaged petri dishes complete medium with the concentration of 1?106?mL-1,and cultivated for 14 d. The expressions of the endothelial cell-specific components ecNOS and flk-1 were detected with RT-PCR; the cells were stained with acLDL-Dil and FITC-UEA-1; the PCDNA3.0-HIF1-? plasmid was transfected into the EPCs mediated by LipofectamineTM 2000,the transcription of HIF1-? was determined with RT-PCR; the expression of VEGF was detected with enzyme-linked immunosorbent assay (ELISA); the expression of HIF1-? was detected with Western blotting.Results After cultivated for 5 d,some round monocytes transformated into spindled inherent EPCs;7 d later,there were some cell colonies containing dozens of cells;10 d later,there were obvious cell clones; RT-PCR showed ecNOS band in 548 bp,flk-1 band in 819 bp; acLDL-Dil and FITC-UEA-1 double-staining was positive; RT-PCR showed specific band in 383 bp; ELISA showed the content of VEGF in the supernatant of EPCs transfected with HIF1-? was (20.53?2.33) ?g?L-1,and it was (3.96?1.67) ?g?L-1 in the EPCs transfected without HIF1-?,there was significant difference between two groups (P