A Study Of Cultured Bone Marrow Endothelial Progenitor Cells Transfected With The Gene Of Human Hypoxia Inducible Factor-1α (HIF1-α) In Vitro

Recent studies found endothelial progenitor cells in the vascular in the peripheral blood, which derived from bone marrow.If the tissue was in the ischemia circumstances, endothelial progenitor cells can be mobilized from the bone marrow, migrate and integrate to the active site of angiogenesis, proliferate and differentiate into mature endothelial cells, participate in the formation of the local neovascularization, the process is known as angiogenesis. At present, adult peripheral blood, bone marrow, cord blood is found to contain the endothelial progenitor cells. HIF-1 is a transcription factor which exist in mammals and hunan under hypoxic conditions, regulate the expression of a variety of target genes such as VEGF, RBC growth factor (EPO), and so on. In our expeiment,we transfected HIF gene into the endothelial progenitor cells, by combining gene therapy with EPCs, on the one hand EPCs can improve the"efficiency", on the other hand, the gene find suitable"vector"to enters the human body to achieve stronger therapeutic effect.This study separated the endothelial progenitor cells by the method of density gradient separation in the human bone marrow, detected the expression of the endothelial cell-specific components --ecNOS, flk-1 (KDR) by the method of RT-PCR, detected the changes of the activity of the NOS in the medium by the method of nitrate reduction, indirectly confirms the function of endothelial cells; stained the cells with acLDL-Dil and FITC- UEA -1, identified by the confocal laser scanning microscopy, confirmed that we isolated and induced endothelial progenitor cells from human bone marrow successfully.We transfected the PCDNA3.0-HIF1αplasmid into the endothelial progenitor cells mediated by LipofectamineTm2000, detected the transcription of the HIF1αby the method of reverse polymerase chain reaction (RT-PCR); detected the expression of VEGF by the method of enzyme-linked immunosorbent assay (ELISA), confirmed that the VEGF levels in the supernatant of endothelial progenitor cells increased by about five times in the transfection team than that in the non-transfected team; detected the expression of the HIF1αby the method of immunoblotting (Westem blot), and ultimately confirmed that PCDNA3.0-HIF1αwas transfected into the endothelial progenitor cells successfully.In the next phase of our study, we will explore the myocardial vascular repair capacity and angiogenesis of the endothelial progenitor cells transfected with HIF gene, provide an alternative programme for the promotion of in heart tissue lesions and find a new ways proceed to repair ischemic myocardial cell Angiogenesis in cell level, create a new approach for the Cell biological treatment of myocardial infarction cells.