| ObjectiveMononuclear cells were isolated from rats bone marrow, then used special Medium make them differentiating to endothelial progenitor cells(EPCs); and hepatocyte growth factor (HGF) gene were transfected into primary cultured endothelial progenitor cells (EPCs) of rats in vitro. Then the HGF protein secreted in culture medium were measured and studying its effect on the proliferation migration and angiopoiesis of EPCs.Methods1. Bone marorw mononuclear cells(BMMNCs) were obtained from bone marrow of 4-6w Wistar rats (120-150g) by flushing rat's femur and tibia and were isolated by density gradient centrifugation.cultured in endothelial growth medium-2 supplied with recombined human VEGF,IGF,b-FGF,FBS and ascorbic acid (EGM-2MV). The cells were observed under inverted microscope every day. The cultured cells were identified by being observed the experiment of uptaking DiL-acLDL and combining FITC-UEA-1 by fluorescence microscope. Also Flow Cytometry was performed to analyze the expression of CD 133^ flk-1,VE-cadherin(CD144) and CD31 dynamically.2. Extraction and amplification the plasmid of pCMV- HGF, To defective adenovirus (Ad-GFP) as a carrier-mediated HGF gene transfer of endothelial progenitor cells and transfection efficiency were identified. To demonstrate the secreted HGF protein concentration in the supernatant by Enzyme-Linked Immunosorbent Assay (ELISA),and study its effect on the proliferation of primary cultured EPCs with the methods of MTT;Transwell cell migration detection; Three-dimensional cell culture assay angiogenesis ability in vitro.Results1. Rat EPCs were isolated and cultured successfully in vitro and were identified by the function of cells and Flow Cytometry;2. HGF were transfected into primary cultured EPCs successfully, green fluorescent protein(GFP) were observed through fluorescence microscope.And 22.15■3.77 ng/ml39.42±7.32ng/ml99.09±9.89ng/ml,311.87±26.56ng/ml,224.72±20.91 ng/ml (ng/ml) HGF protein were detected in culture medium after EPCs were transfected 1d,2d,4d,7d and 11d respectively. Furthermore, the HGF gene has the ability to stimulate the proliferation of EPCs. After transfected HGF gene, EPCs could promoted the proliferation.The proliferation of HGF groups were significantly greater than that of control group from 4d to 7d (P<0.05) except that from 1d to 2d, Migration of transfected cells increased; three-dimensional culture to form tubular structures visible.Conclusions1. Rat BMMNCs were isolated from rat bone marrow and differentiated to endothelial progenitor cells successfully in vitro.2. Mediated by defective adenovirus can be successfully HGF gene transfer into vascular progenitor cells, and in the culture supernatant of HGF protein could be detected, confirmed that the transfected gene can be effectively expressed in cells And to promote EPCs proliferation, migration and angiogenesis,which provides the support for further cure of the limb ischamia disease with gene-stem cell therapy. |
PDF Full Text Request