Experimental Study Of Endothelial Progenitor Cells From Human Cord Blood Transplantation Into Myocardial Infarction In Nude Mice

Myocardial cells are considered as a kind of terminal differentiated cells in traditional concept, myocardial cells show a large number of necrosis when the myocardial cells during ischemia in the early, it causes cardiac systolic dysfunction; the post-necrotic myocardium is replaced by fibrous connective tissue, non-ischemic regional cardiac eccentric hypertrophy further lead to reduced myocardial function. The reconstruction of coronary blood supply is the most effective and fundamental way to treat MI, and technology of the cell transplantation develops a new way in recent years.Cell-mediated vascular regeneration discussed in this paper can preserve heart function and improve prognosis of myocardial infarction by re-constructing blood supply to the ischemic tissue. The experiments were designed to prove whether endothelial progenitor cells can be isolated and cultured from human cord blood under certain culture conditions, which transplanted into ischemic myocardium of nude mouse. To obverse the generated new capillaries to improve heart function, and to investigate the possibility of endothelial progenitor cells transplantation for the treatment of myocardial infarction of nude mouse. Part one Culture of mononuclear cells and characterizations of endothelial progenitor cells from human cord vein bloodObjective:Mononuclear cells were separated from fresh human cord vein blood, it was cultured within the certain inductors. The attached cells were identified and confirmed as endothelial progenitor cells.Methods:1. The taking of human cord vein blood:After pregnant women and their families authorized, the five cases caesarean section of healthy full-term pregnant women were selected randomly. Heparin was prepared with the concentration of 50u/ml, cord blood was immediately collected with anticoagulant syringe under sterile conditions after the placenta peeling after stripping.40ml blood was completely mixed with heparin and processed within 2 hours.2. Separation of mononuclear cells from human cord blood:The human cord blood diluted with the same volume DMEM medium. Diluted human cord blood was carefully tiled on an equal volume of the Bioshop lymphocyte separation medium at 2000 r/min under the centrifugal 20min. The liquid in centrifuge tube was divided into three layers, the upper level was plasma, most of the lower were red blood cells and granulocyte. The middle was lymphocyte separation medium, in the upper and middle layer was a narrow band of white cloud mainly consisting of mononuclear cells. Sucked mononuclear cells and adding five times the volume of no-serum DMEM at 1500r/m under the centrifuge for 10min, finally wash cells again.3. Inducing conditions:The mononuclear cells were application of DMEM medium and 20% fetal calf serum, which added within the vascular endothelial growth factor (VEGF165,20 ng/ml),basic fibroblast growth factor (b-FGF,1 ng/ml), Insulin-like growth factor-1 (IGF-1,2 ng/ml), glutamine (300μg/ml), penicillin G (100U/ml) and streptomycin (100mg/L). The mononuclear cells were inoculated on gelatin-coated 6-hole culture plates at density of 5×105/ml. Changing the fluid for the first time at the 3rd day, thereafter every 2-3 days, before changing the liquid and recording the cell morphology with the inverted microscope.4. Detection indicator:The 7th adherent cells were contained and cultured in Dil-acLDL (10μg/mL) at 37℃in dark for 4 h, and then in 4% neutral formaldehyde for 10min, PBS rinsing fluid. Added FITC-UEA-1 (10μg/mL) and incubation in dark for 1h. PBS rinse fluid again and the cells were observed under fluorescence microscope after the cells staining. Two kinds of positive staining cells were considered as EPCs. At 7th days, CD34 and CD133 surface molecular expressions were investigated by flow cytometry, RT-PCR studies with cultured cells from the existence of VEGFR-2 mRNA.Results:1. EPCs cell morphological changes:MNCs were separated with the density gradient centrifugation, more than 95% of the cells were alive by trypan blue staining. After MNCs cultured with these conditions, the characteristics of cells were observed under the microscope. MNCs was circular and evenly distributed suspension. Discarded the non-adherent cells after the 3rd day, a few small spindle cells were seen. MNCs were round cells and similar to the white blood cells in the early stage, but the round cells decreased and appeared a small colony after the 7th day. The cultured cells showed spindle-shaped cells or multi-protruding cells, a large part of the cells division phase can be seen in these cells with high-power microscope. Colonies evolved a typical colony after about the 14th day and spindle cells gradually shortened, finally the typical characters of endothelial cells showed as cobblestone-like change. 2. By flow cytometry:Take attached cells on the 7th day, CD34 and CD133 positive rate (50.48±5.17)% and (19.12±4.37)% by flow cytometry.3. Fluorescence microscopy:more than 90% of the attached cells were able to uptake both DiI-ac-LDL and FITC-UEA-1.4. RT-PCR detection:RT-PCR prompted VEGF receptor-2 of the attached cells was positive.Conclusions:We applied gradient centrifugation to separate mononuclear cells from human cord blood which were inoculated and cultured on gelatin-coated plastic plate with VEGF, bFGF and IGF-1 existence of medium. Although these cells were still mixed with hematopoietic cells, endothelial cells and so on in the early, but more pure cells were obtained with further cultured. EPCs had a number of surface molecules, the application of commonly-used international EPCs identification were identified in this experiment. By the 7th day, identification of flow cytometry which showed expression of CD34 induced of its positive rate of (50.48±5.17)%, expression of CD133 for (19.12±4.37)%, more than 90% of two kinds of attached cells staining positive, and can express VEGFR-2. All these suggested that the method can be simply and rapidly inducted MNCs to EPCs.Part two Experimental study of endothelial progenitor cells from human cord blood transplantation into myocardial infarction in nude mice.Objective:Immune function of EPCs from human umbilical vein is not yet mature, and immunogenicity of weak and graft-versus-host disease incidence is low, sub-HLA loci is high tolerance when transplanted. The adherent cells, which were inducted from mononuclear cells in vitro for 7 days, were transplanted as progenitor cells into nude mice that established a myocardial infarction model, the use of nude mice immune defects observed endothelial progenitor cells in human umbilical cord blood transplanted into nude mice infarct area. Effects of cell transplantation confirmed by infarct area neovascularization, which explored the effectiveness of myocardial ischemia.Methods:1. Grouping:20 female BALB/c-nu nude mice were randomly divided into experimental group and control group (n=10).2. Animal model:Each nude mouse was anesthetized by intraperitoneal injection of 10% solution of chloral hydrate. Each 0.05ml if with a poor effect, added 0.02ml. Connected 16-channel physiological recorder (PowerLab, Australia ADInstruments company) to record ECG. Tracheal intubation with 24G intravenous catheter needle, constant volume model maintained respiration with small animal ventilator (DH-150, Zhejiang University),respiratory rate 90 times/min, tidal volume of 3-6ml, inspiratory:expiratory ratio of 1:1, the success of tracheal intubation was confirmed with there thorax flattering and lungs can be seen through the skin expanded well. Cuting midline of sternum about lcm, pulling the chest wall with clamps to expose the heart. Gently fixed the heart with non-tooth forceps, and ligation the left coronary artery between vertebral arteries and the left atrial appendage with 7-0 Prolene line, and then observed the color changes in left ventricular and record changes in electrocardiogram. After left ventricular changed white, and ECG ST-segment elevation shall be ligated successfully, turn a little large tidal volume before closed the chest.3. Treatment:On the 7th day, the attached cells cultured with CM-Dil (2μg/mL) at 37℃in dark 5min, then placed in 4℃refrigerator, after 15min later the cells were collected and centrifugated, the cells concentration of 107/ml prepared. The experimental group injected 0.3ml EPCs labeled CM-Dil in the tail vein, the control group injected directly DMEM medium.4. Observe the results:After 2 weeks later, the nude mice were killed and the heart was frozen in liquid nitrogen, made 1-2mm thick slices with the direction of long axis perpendicular after 30min in order to calculate MI area with TTC staining method. The remaining specimens were frozen sections (thickness of 5μm), new capillaries and the fluorescence of myocardial infarction was observed under fluorescence microscope in the frozen section. The experimental group and control group parts of the histological changes in myocardial infarction were observed under light microscope. Five vision in the infarct border zone of each slice were randomly observed under the high power field (×200), calculated the average number of capillaries for each slice.Result:After 2 weeks, the experimental group survived seven nude mice and six in the control group, the survival rate of myocardial infarction was 65% in this experiment.1. Infarct area:Nude mice were sacrificed after 2 weeks, pathological visible capillaries and fibroblast cell growth can be seen in the myocardial infarction region; calculating myocardial infarction area, the experimental group was (8.27±1.64)% and (14.30±2.84)% in control group, it was statistically significant difference between the two groups (t=-4.78, P=0.001).2. Capillary density:Counted of capillary density after 2 weeks, the average density of new capillaries per high power field was 14.29±1.38 in the experimental group, control group was 10.17±1.72, it was statistically significant difference between the two groups (t=4.79, P=0.001).3. Fluorescence of new capillaries in infarct area:Nude mice were sacrificed after 2 weeks later, red fluorescence of new capillaries in infarct area can be observed in experimental group under fluorescence microscope,, while the control group were negative.Conclusion:Because of small type, cardiac function of nude mice is difficult to detect directly or indirectly, cardiac function was indirectly responded by counting the MI size and capillary density in this experiment. The average infarct size and capillary density in the experimental group were statistically significant compared with the control group in the experiments. In addition, CM-Dil was often used as a fluorescent tracer, EPCs uptake CM-Dil were transplanted into nude mice in this experiment, the new capillaries were observed the red fluorescence under fluorescence microscope after two weeks, which confirmed that EPCs participated in the new capillaries. The mechanisms of beneficial cardiac function for EPCs transplantation remain to be further substantiated, it may generate new blood vessels, or differentiate into mature endothelial cells,or secret a variety of vascular growth factors to promote the host autologous myocardial angiogenesis, which improve blood supply by reconstructing infarction area.Nude mouse model of myocardial ischemia were established in this study, and mononuclear cells from human cord blood, which differentiate into endothelial progenitor cells in vitro, were observed that can reduce the myocardial infarction area and increase in capillary regeneration after transplantation on myocardial ischemia. And indirectly confirming transplantation the endothelial progenitor cells from human cord blood had a therapeutic effect on myocardial ischemia.