Endothelial Progenitor Cells(EPCs) Derived Rat Bone Marrow Isolated, Cultured, Diferentiated And Qualificated

Objective To explore the procedures of isolated, cultured, diferentiated andqualificated from rat bone marrow-derived endothelial progenitor cells and provide theplatform for the experimental study and the clinical practice.method Bone marrow mononuclear cells(BMMNCs) were harvested from bonemarrow by flushing rat femur and tibia and were isolated by density gradientcentrifugation, cultured in endothelial growth medium-2 supplied with recombined humanVEGF, IGF, b-FGF, FBS and ascorbic acid. The cells' morphology were observed bymicroscopy. MTT assay was used to study the activity of proliferation. Alsoimmunocytochemistry and immunofluorescence were performed to analyze the expressionof CD34, VEGFR-2, vWF, and progentitor cell marker CD 133 dynamically. Observed theexperiment of uptaking DIL-AC-LDL and combining FITC-Lectin-UEA-1 by laserscanning confocal microscope, of qalificating the ultramicro-structure and endothelial cellscharacteristic(Weible-Palade body) by transmission electron microscope (TEM) andanalysed the chromosome karyotype as to safety evaluation.Result The lately-separated bone marrow mononuclear cells (BMMNCs) presentcircular and the size is different. Parts of the cells start attaching after 48 hours. Themorphology formed the shuttle, triangle, the spindle or irregular form. The attached cellswere able to form cells'colonies and line up in the core-like structure from 4 to 8days, and get close to coalesce and exhibite the typical cobblestone morphology from 9 to11 days. Cell growth curve was typical "S" shape. There were significant differences in theproliferation activity day 8 and day 10 compared with that of day 5 and day 14 inculture. The attached cells were positively stained for CD34, VEGFR-2, vWF and CD 133and change dynamically, had uptaken DIL-AC-LDL and combined FITC-Lectin-UEA-1, observed the characteristic Weible-Palade body, and be genetically stable.Conclusion The experiment had set up a preliminary system of endothelial progenitor cells derived rat bone marrow isolated, cultured, diferentiated and qualificatedin vitro.