| Research Background:The research about cells transplantation for the treatment of heart disease especially ischemic myocardium is becoming an increasingly promising strategy. Stem cells transplantation for the treatment of damaged myocardium after myocardial infarction may transdifferentiate into cardiomyocytes and blood vessels give people new hope. Stem cells have the potential to enhance myocardial perfusion and/or contractile performance in patients with acutemyocardial infarction, advanced coronary artery disease, and chronic heart failure. The dogma of the heart as an organ composed of terminally differentiated myocytes incapable of regeneration is being challenged. Evidence has been presented that a fraction of cardiomyocytes may be able to reenter the cell-cycle and that limited regeneration can occur through recruitment of resident and circulating stem cells. Stem cells are capable of self-renewal, transformation into dedicated progenitor cells, and differentiation into specialized progeny. Moreover, the mechanistic underpinnings of stem cell therapy appear to be far more complex than previously anticipated. The cell surface antigen CD133 is expressed on early HSCs and EPCs, both of which collaborate to promote vascularization of ischemic tissues. CD133~+ cells can integrate into sites of neovascularization and differentiate into mature endothelial cells.Research Objectives:To study biological traits of CD133~+ cells from human umbilical cord blood were isolated and induced to differentiate. To set up a practical method for purification of CD133~+ stem cells. To improve the method to estabilish the model of ischemic myocardium in nude mouse heart, and improve the successful rate, and ensure that the experiment can be performed successfully. To investigate the proliferation and differentiation of CD133~+ cells after being implanted into ischemic myocardium, and the reduction of theinfarction and fibrosis in ischemic heart of mouse. Methods and Results:Firstly, we have set up a technical platform for isolation, expansion, purification and identification of CD133+cells from human umbilical cord blood, to study biological traits and differentiate of CD133+cells from human umbilical cord blood, to set up a practical method for purification of CD133+stem cells. CD133+ and CD133 cells selected from fresh cord blood were cultured in StemSpan? SFEM serum-free medium supplemented with SCF, IL~3, IL-6. Attached spindle-like cells were detached and labeled with a series of antibodies against endothelial-specific markers. After the immmunomagnetic separation the yield of CD133+ cells was (6. 08~ 6. 80) X105cells/ml; The purity of isolated CD133+ cells was (96. 18±1.83) %. Attached spindle-like cells appeared after 4 days culture and formed cord-like structure by 15 days. These cells can bind UEA-1 and express endothelial-specific markers, including vWF. After Ficoll-Paque density centrifugation and the immmunomagnetic separation, we can get high purity CD133+ and CD133 cells, which can differentiate into endothelial cells.In the second part we improve the method to establish the model of ischemic myocardium in nude mouse heart, and improve the successful rate, and ensure that the experiment can be performed successfully. The nude mouse (age: 4-6W, weight: 20~25g) were usedto make the model of ischemic myocardium. The ribs of mouse were cut off after arterial intercostals were ligated. The thorax dilater was not used, and instead, the stump of ribs was extracted to the opposite sides with wire ligature to expose the heart. The electric coagulation of coronary artery was performed in the thoracic cavity. The successful rate of model improvement method was 70%, and that of traditional method was 50% . The improved method is simple and convenient, with high successful rate.The aim of third part was to experimental study on the effect of cord blood CD133+ cells transplantation into the ischemic myocardium in nude mouse. CD133* cells selected from fresh cord blood were cultured in vitro and labeled with PKH26. The CD133+ cells were then transplanted into the ischemic myocardium whose left descending branch of coronary artery had been coagulation (group I). The rats receiving serum-free medium injection were used as controls (group II). Two weeks after transplantation, the infarction and fibrosis in ischemic heart were measured. The infarction and f ibrosis were reduced two weeks after transplantation of CD133+ cells in group I compared with that in group II (K0. 05). The CD133+ cells injections could reduce the area of infarction and fibrosis in ischemic heart of nude mouse.Conclusions:1. After Ficoll-Paque density centrifugation and theimmmunomagnetic separation, we can get high purity CD133*...
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