| Coronary artery disease has currently become one of the major life-threatening cardiovascular diseases in China. Although many advances, such as intra-vascular thrombolysis, angioplasty, and coronary bypass grafting, have been achieved on the treatment of ischemic heart disease, the prognosis of severe diffused ischemic heart disease has not been improved. It has gained more and more attention on how to recover the blood supply to the ischemic regions and how to rebuild myocardial structure of severe diffused ischemic hearts. The experiment was designed to prove whether endothelial progenitor cells can be isolated and cultured from cord blood under certain culture conditions, and whether neonatal peripheral mononuclear cells, which have the same components as cord blood, can alleviate left ventricular remodeling after transplanted into ischemic myocardium.Part one Culture and characterizations of cord blood mononuclear cellsObjective: To investigate whether endothelial progenitor cells can be isolated and cultured from cord blood.Methods: Cord bloods were collected and mononuclear cells were isolated within 2 hours. Mononuclear cells with a density of 5×105/ml were cultured in DMEM medium with 20% fetal bovine serum and proper factors as supplement Growth statuses of cells were observed and growth curve was made by MTT method. At 10 days, KDR, VE-cadherin, CD34 and CD45 surface molecular expressions were investigated by flow cytometry and uptake of acetylated-LDL was analyzed by fiuoscent microscopy. RT-PCR was performed to detected eNOS and FLT-1/VEGF receptor-1 mRNA.Results: CD34 positive ratios were 3 ± 0.6% for adult peripheral bloods and 22 ?4% for cord bloods. After 48 hours, spindle-shaped cell sprouted from attached cells. At early phase, round shaped cells, which resembled leucocytes, could be observed, but the number of these cells decreased at late culture phase. At 10d, cultured cells showed spindle or polygon shapes. Some of the cultured cells grew in cord-like shape, and many of these cells were observed in dividing phase of cell cycle. AT cells expressed KDR, VE-cadherin, and CD34 but not CD45. More than 95% of attached cell could uptake Dil-ac-LDL with red fluorescence. After RT-PCR, Amplified eNOS and FLT-1/VEGF receptor-1 products were detected.Part two Study on Cell Viability and Impact on Left Ventricular Remodeling after Neonatal Mononuclear Cells Transplanting into Ischemic MyocardiumObjective: To investigate the validity of cell transplantation in the treatment of acute myocardial ischemia by neonatal dog peripheral mononuclear cells, which have the same composition and characteristic as cord blood counterparts.Methods: 16 mongrel dogs were divided into test and control groups randomly. Neonatal dog peripheral bloods were collected and mononuclear cells were separated by gradient centrifuge. PKH26 fluorescent dye was used to trace the transplantedcells. Left descending arteries were ligated at the distal side of the second diagonal branch. 1 hour later, mononuclear cells were injected into peri-ischemia regions with total number of 108 at 25 points, 0.04ml per point. Non-serum mediums were injected into ischemic myocardium of control group. At day 2 and day 30, trans-thoracic echocardiography (SONOS 5500) with S4 probe were undertaken to calculate heart function index and to investigate left ventricular morphology. Quantitative regional systolic and diastolic functions were analyzed with doppler tissue imaging. The sizes of ischemic region were calculated. Vascular intensity and collagen were studied in heart specimen.Result: PHK26 labeled cells were detected in the mononuclear cells transplanted myocardium. These cells incorporated into vascular network of ischemic tissue. Necropsy examination disclosed that capillary density was significantly greater in test group than in the control group. Moreover, the extent of left ventricular scarring was significantly less in dogs receiving monoclear cells than in controls. Left ventricular morphology changed af...
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