| Objective: To observe the treatment possibility and effects on rats models with experimental femoral artery obliterans by transplantation of endothelial progenitor cells(EPC) from peripheral blood mononuclear cells and bone marrow mesenchymal stem cells(MSCs).Methods: Experimental rats models with both femoral arteries obliterans were successful made by dissecting ,ligating, and excising both femoral arteries as well as their branches in 28 rats.Our study consisted of 2 parts.Study 1: Auto-transplantation of mononuclear cells from rats' peripheral blood. Peripheral blood was withdrawed by cardiopuncture in 12 rats' models, the mononuclear cells were isolated by density gradient centrifugation under sterile environment. 1ml mononuclear cells(0.5~2 107/ml) were auto-transplantated into right hindlimbs by intramusculer injection as group A1 in 12 rats' models, and the same volume of normal saline as control were similarly injected into the opposite hindlimbs as group B1. Laser Dopplerperfusion imaging was taken at 0 day, 14 day and 28 day after transplantation respectively. On the 28 day after transplantation,all rats were sacrificed and their muscles in AI and BI were examined by microscopy for immunohistochemical and histological examinations. Study 2, Allogeneic transplantation of endothelial progenitor cells from bone marrow. MSCs were isolated from allogeneic male Wistar rat, then were cultured in DMEM including 10% fetal bowine serum(FBS) at 37 C,5% CO2 circumstance in vitro. EOF, bFGF and IGF-1 were added into culture medium at 10th day and the active endothelial progenitor cells were labled with BrdU. 5 106 of above cells were transplanted into right hindlimbs by intramusculer injection as group A2in 16 rats of experimental femoral obliterans models, and same volume of normal saline were injected into the opposite hindlimbs as group B2 as control. Laser Doppler perfusion imaging and 28 day, 8 rats were sacrificed respectively and muscles of alwas taken at 0 day, 14 day and 28 day after transplantation. On 14 day 1 hindlimbs were extracted for immunohistochemical examinations(by F V1I1 and BrdU).Results: Study 1:(1)In group A\ the skin blood perfusion in hindlimbs were significantly increased as compared with group B1: at 14d after transplantation the blood perfusion in group A1 :2.34 0.26V , group B1: 2.06 0.19V, P=0.0216; at 28d group A1: 2.6 0.16V, group B1: 2.12 0.15V, P=0.0007. (2) The numbers of blood vessels with positive staining of Factor VIII in hindlimb at 28d after transplantation in group A1(8.86 1.59) were obviously more than that in group B1(5.46 1.06) (P=0.0000).Study 2: (l)In group A2 the skin blood perfusion in hindlimb were significantly increased as compared with group 82: at 14d after transplantation the blood perfusion in group A2: 2.43 0.29V, in group B2: 2.04 0.19V, P=0.0124; at 28d in group A2: 2.81 0.22V, in group B2: 2.11 0.17V, P=0.0003. (2) Some positive stained endothelial cells by BrdU were found in A2 but not in B2. (3)The numbers of blood vessels with positive staining of Factor VIII in hindlimb at 28d after transplantation in group A2(12.1 2.47) were obviously more than that in group B2(5.4 0.97) (P=0.0000).Conclusion: Transplantation of EPC from both peripheral blood mononuclear cells and from cultured MSCs in vitro can significantly increase the skin blood perfusion and capillary density in ischemic hindlimbs in rats with experimental femoral artery obliterans as compared with that in control hindlimbs. The BrdU positive staining in capillary in group A2 may suggest that the EPC which we transplanted into ischemic tissue can improve and increase the angiogenesis. The EPC may come from the mononuclear cells of peripheral blood or from bone marrow cells.They have potential to differentiate into vascular endothelial cells in ischemic tissue in vivo.The differentiation and development potentiality of EPC to vasoendothelial cells in ischemic tissue was much better than that of naturally auto-angiogenesis in vivo.
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