Treatment Of Experimental Femoral Artery Obliterans In Rat By Transplantation With Bone Marrow Mesenchymal Stem Cells From Bone Marrow

Objective:Study rat Marrow Mesenchymal Stem Cells(MSCs) differentiating into Endothelial Cells under certain condition and treatment of experimental femoral artery obliterans in rat by transplantation with MSCs only or MSCs with necessary inductor.Methods:1. MSCs were isolated from healthy rat and purifided by density gradient centrifugation, collect the Mononuclear cells(MNCs) layer. All cells were seeded into six-pore plaque containing Dulbecco's-modified Eagle's Medium-Low Glucose (DMEM-LG), penicillin 100 U/ml, streptomycin 100μg/ml, and 10% fetal calf serum (FCS). MSC cultures grew at 37°C in 5% CO2. Nonadherent cells were removed after 24 hours. The medium was changed subsequently every 2 days. Two weeks later the culture reached 90% confluency. MSCs were recovered using 0.25% Trypsin-EDTA and replated at a density of 5,000–6,000 cells per cm2 of surface area as passage 1 (P1) cells .When the amount of MSCs reached 1×10~7cells , special antigen of CD34 and CD29 was tested by flow cytometry.2. Confluent cells(MSCs)from passages P4 were cultivated in six-pore plaque containing DMEM, 2% FCS or 10% FCS, 10 ng/ml VEGF, 2ng/mlbFGF. Medium was changed every 4 days. When culture reached 90% confluency., 0.25% Trypsin-EDTA was used to recover and replate them. Special antigen of FVIII and CD34 was tested after 7 days and 14days.3. Experimental rats models with both femoral arteries obliterans were successful made by ligating, and excising both femoral arteries as well astheir branches in 18 rats. In 6 rats' models, 1 ml A fluid (normal saline) as control were injected into left hindlimbs by intramusculer injection as group A and the same volume of B fluid (DMEM, 1×106cells/ml MSCs labled with BrdU ) were similarly injected into the opposite hindlimbs as group B. Another 6 rats' models, 1 ml C fluid(DMEM, 1×106cells/ml MSCs labled with BrdU, VEGF and bFGF ) were injected into left hindlimbs, 1 ml A fluid injected into right hindlimbs. The last 6 rats' models, B fluid were similarly injected into left hindlimbs, 1 ml C fluid injected into right hindlimbs. 7 days and 14 days later, Hindlimb muscule was observed in sections stained with hematoxylin and eosin, FVIII was investigated using immunohistochemical methods.Results:1. Phase contrast microscopy from cells after cultivating 4 days demonstrated a fibroblast-like, spindle-shaped morphology. In later 10 days, the spindle-shaped cells began to display a broadened, flat morphology, and arrange like a group of fish. FCM(flow cytometry) analysis showed that 97.32±2.77% cells expressed MSCs typical antigens CD29,1.01±0.56% cells expressed CD34 which is a antigens of Hemopoietic stem cell.2. Immunohistochemical staining of FVIII showed that 12.21±2.32% cells in 10% FCS expressed Endothelial Cells typical antigens FVIII after 14 days, but 91.43±6.32% cells in 2% FCS expressed Endothelial Cells typical antigens FVIII.3. We introduced differentiation into endothelial-like cells by cultivating confluent MSCs in the presence of 2% FCS and 10 ng/ml VEGF, 2 ng/ml bFGF for 7 days and 14 days , FCM and immunohistochemical methods was used to test CD34 and FVIII, the result was shown in table 1. But cell morphology showed no difference compared with undifferentiated MSCs. Table 1. the expression of CD34 and FVIII in different times4. After 7 days some positive stained endothelial cells by BrdU were found in group B and C but not in A. After 14 days, blood vessels with positive staining of Factor VIII in hindlimb were found in group B and C but not in A. The numbers of blood vessels with positive staining of Factor VIII in hindlimb at 14d after transplantation in group C(12.6±2.9) were more than that in group B(8.4±2.3) (p<0.05), and group B obviously more than that in group A(2.8±0.6) (p<0.01).Conclusion:1. MSC is another kind of marrow stem cell and have ablility of differentiation into Endothelial Cells under certain condition.2. MSCs differentiating into Endothelial Cells in 2% FCS were better than that in 10% FCS.3.Transplanting of MSCs from cultured MSCs in vitro can significantly increase the amount of blood vessels in ischemic hindlimb, thus improve the blood circulation of ischemic hindlimb.4. Transplanting of MSCs or MSCs with necessary inductor can obviously improve and increase the angiogenesis. But transplanting of MSCs with necessary inductor was better than MSCs only.