Transplantation Of Endothelial Progenitor Cells Labeling With New Superpara-magnetic Iron Oxide In The Treatment Of Deep Venous Thrombosis

Objective: To research bone marrow-derived endothelial progenitor cells (EPCs)labeled with new superpara-magnetic iron oxide (SPIO) were transplanted to the model ofdeep venous thrombosis in the rat, tracking stem cells in vivo, which provided a noinvasiveand effective monitoring technology for the treatment of deep venous thrombosis.Methods: EPCs were extracted and purified from rat bone marrow by density-gradient centrifugation and the method of adherence-sieve, and then incubated with sixdifferent concentrations of SPIO particles(0ug/ml、6.25ug/ml、12.5ug/ml、25ug/ml、50ug/ml、100ug/ml), which was made up with Fe3O4embellished by APTS. we exploredthe labeling efficiency and the effects on cell proliferation and cell activity of SPIO byusing Prussian blue staining, Trypan blue staining,Transmission electron microscopy,MTT test and flow cytometry, and confirmed the best labeling concentration and time.Experimental rat model of deep vein thrombosis were obtained by embolization withgelatin sponge the inferior vena cava. The rats were randomly divided into four groups,each group have ten model rats (A group：1ml106EPCs labeled with SPIO transplantation;B group:1ml106EPCs labeled with Dil transplantation; C group:1ml106simple EPCstransplantation; D, control group:1ml medium (EGM-2MV) transplantation. The7th,14th,21th,28th day after transplantation, we were planning to use MRI to observe the biologicalproperty, such as migration, homing, differentation. Meanwhile, HE staining andImmunohistochemical staining was performed to detect recanalization and theneovascularization. The capillary density was determined quantitatively by counting thecapillaries under high-power microscope. The SPSS17.0software used for analysis,P<0.05for the difference was significant.Results: Bone marrow-derived EPCs can be successfully isolated and cultured by Density-gradient centrifugation and adherence-sieve. Prussian blue staining showed thatSPIO was up taken by EPCs in a concentration dependence manner. There is the highestand best labeling efficiency about90%Prussian blue staining positive cell when theconcentration of SPIO is25ug/ml, about60%when the concentration of SPIO is50ug/ml,and the number of living cell is under20%when the concentration of SPIO is100ug/ml, itis the best banlance between the labeling efficiency and cell viability when theconcentration of SPIO is25ug/ml, showing a significant difference (P＜0.05).Transmission electron microscopy showed the ultrastructure of EPCs was well preserved.Abundant SPIO granules were found in organelles, such as lysosome, smooth endoplasmicreticulum and mitochondrion. MTT test and flow cytometry showed that cell viability,proliferation and differentiation ability of labeled cells were not affected, and the labeledcell can be used stem cell tracer in vivo. The MRI showed EPCs had been homing andmigrating to the thrombosis in the inferior vena cava and was seen high density briquettingshade. We observed a mass of the new capillary in thrombosis after recanalization and theneovascularization by HE staining and Immunohistochemical staining. All these newcapillaries expressed the specific antibody CD34and vWF belonged to the endothelialcells.Conclusion: Bone marrow-derived EPCs can be successfully isolated and cultured byDensity-gradient centrifugation and adherence-sieve, and easily and efficiently labeled bySPIO without interference on the cell viability and proliferation, which lay the foundationsfor the further stem cell tracer in vivo and provided a new research approach for thetreatment of deep venous thrombosis.