Treatment Of Lower Limbs Ischemia By Transplantation With Endothelial Progenitor Cells From Human Umbilical Cord Blood

[Objective]To investigate the effect of transplantation endothelial progenitor cells which proliferated and stimulated from human umbilical cord blood on ischemically bind limb in nude mice.[Methods]1.The umbilical cord blood of newborns was collected from the umbilical cord vein of healthy mother.Human umbilical cord blood mononuclear cells were isolated by Ficoll density-gradient centrifugation, then plated on dishes coated with human fibronectin.After 2 days of culture, the nonaderent cells were collected and replated onto fibronectin-coated dishes.The cells were observed and identified with the techniques of immunohistochemistry, immunofluorescence and flow cytometer after 7 days culture.2.Endothelial progenitor cells were digestived and collected after 7 days. Cells were plated into 24 hole training board and cultured at different cell densities(1×10³,1×10⁴,1×10⁵/cm²)and in basic medium(M199,EGM-2,DMEM)supplemented with FBS(5%,10%,20% ) and different combinations of cytokines ( VEGF,VEGF+bFGF,VEGF +bFGF+EPO), the experiment was based on L₉(3⁴) orthogonal design. All groups of cells were centrifuged after 7 days, we selected the best culture condition which expand endothelial progenitor cells. All cells were identified with flow cytometer, immunofluorescence and angiogenesis function.The migration function of EPCs was determined using modified Boyden chamber.Four groups were determined according to SDF-1 concentration: control, 10ng/ml, 50ng/ml, 100ng/ml, respectively.After 30 minutes,we compute the cell numbers whitch move to the next room.3.After establishment of unilateral hindlimb ischemia in athymic nude mice, 40 nude mice were divided into 4 group: group A(EPCs),group B(EPCs+SDF-1), group C(SDF-1),group D(M199).We observed skin color of ischemic limbs after transplantation;detected skin temperature;statisticing salvage rate of lower extremity limb;observed angiogenesis in ischemic muscle;analysised transplantation efficiency of EPCs after amplified and treatmented. [Results]1.Attached colony-like growth cells appeared after cord blood mononuclear cells culture 5 days and became spindle shaped at 10 days of culture.After 21 days they differentiated into mature ECs forming cobblestone. Attached cells were stained red by Dil-acLDLab and green by FITC-UEA-1 and yellow by double-positive,and more than 75% adherent cells were double-positive.The positive rates of CD34, CD133, VEGFR-2 andâ…§factor of ex vivo expanded EPCs at 7 days of culture were 51.4%,28.7%,82.8%,80%,respectively.2.Regardless of culture conditions,all cells proliferated.In terms of expansion multiple level,its effect was cytokines>basic medium>FBS concentration>culture density.The expansion multiple was highest (80 times) when culture density was 1×10⁴/cm² and culture medium was M199 supplemented with 5% FBS and VEGF+ bFGF+EPO. After amplification, more than 65% cells were double-positive and the positive rates of CD34, CD133, VEGFR-2 andâ…§factor were 41.6%,23.6%,88.3%,85%,respectively. Cells incubated in ECMatrix^TM in vitro can form vascular network-like structure.The number of migration cells when the concentration of SDF-1 was 0ng/ml, l0ng/ml, 50ng/ml, 100ng/ml were 19.33±2.02, 29.33±1.45, 71.66±3.48, 88.66±2.60, respectively. The number of adhesion cells were 29.3±1.52, 44.6±2.5, 74.6±2.51 and 84.0±3.00, respectively. The migration and adhesion activity was significantly higher in group of l0ng/ml, 50ng/ml, 100ng/ml than control group(P<0.05).The difference was determined in comparison of l0ng/ml VS 50ng/ml(P<0.05), 10ng/ml VS 100ng/ml(P<0.05), 50ng/ml VS 100ng/ml(P<0.05),respectively.3. After treatment in nude mice' ischemia hindlimb,the ischemic scores in group A, B, C and D were 1.9, 1.4, 2.4 and 2.5, respectively. The rate of ischemic hindlimb reserving in group A, B, C and D were 40%, 60% , 20% and 20%. Hindlimb'skin temperature decreased in group A and B were more lower than group D(P<0.05), group B were more lower than group A(P<0.05).28 days after treatment, the capillary density in group A, B ,C and D were 3.94±1.26, 5.63±1.22, 2.52±0.87 and 2.37±0.66, respectively. The capillary density was significantly greater in group A and group B vs group D(P<0.05), group B vs group A(P<0.05).There is no difference of capillary density in group C and group D.[Conclusion]1. Sufficient EPCs can be obtained from cord blood through amplification in vitro.The expansion multiple was highest when culture density was 1×10⁴/cm² and culture medium was M199 supplemented with 5%FBS and VEGF+bFGF+EPO.2. SDF-1 promote EPCs migration and adhesion in a dose-dependent manner. SDF-1 augments neovascularization by inducing EPCs recruitment in ischemic organization.3. EPCs can increase vessel density to improve sympotoms of ischemic lowe limbs in nude mice.4. Amplification and stimulated EPCs in vitro can solve the promble of poor numbers of EPCs and improve transplantation efficiency of human umbilical cord blood..