| Advances in the past five decades, including risk-factor modification, use of βblockers and angiotensin-converting-enzyme inhibitors, and effective percutaneous intervention ,have reduced the mortality and morbidity of ischemic heart disease. However, ischemic heart disease accounts for 50% of all cardiovascular deaths and nearly 40% of the heart failure incidents in the world. The progressive nature of ischemic heart disease, restenosis after percutaneous intervention, and some difficulty in gene therapy have exhausted patients and their doctors. Many researchers have investigated cell transplantation and considered it as an alternative treatment for heart disease. Now it is accepted that the adult bone marrow contains mesenchymal stem cells (MSCs), which contribute to the regeneration of mesenchymal tissues such as bone, muscle, cartilage, ligament, tendon, adipose and stroma. So far, we donnot find special marker of MSCs. According to Friedenstein s and Wakitani s methods, we isolated bone marrow mononuclear cells (BM-MNCs) by centrifugation using Ficoll of a density of 1.077g/mL. BM-MNCs contain various kinds of cell lineages, such as mesenchymal stem cells, hematopoietic cells, fibroblasts, and osteoblasts, as well as cells of endothelial lineage. The present study using the infarcted model was designed to test therapeutic effectiveness of MNCs, including the potency of differentiation into cardiomyocytes, improving post-infarcted heart function, and neovascularization.Our study is divived two parts- In vitro and animal implant study. In vitro MNCs were aspirated from 5 months Japanese white rabbits' tibias, isolated by Ficoll gradient(density:l .077g/ml), then treated with l祄ol/L of 5-azacytidine for 24 hours at the third day of seeding. After 20 days, MNCs differentiation was observed by immunohistochemistry staining and electron microscopy. As a result, about 30% cells in culture were found positively stained with cardiac troponin T, a -sarcomeric actin, and connexin-43. And myofilaments can be seen by electron microscopy. In vitro evidence supports that BM-MNCs convert to a myogenic phenotype after with treatment lowdoses of the DNA demethylation agent 5-azacytidine. We proposed that these cells contain a myogenic determination locus in a methylated state with a transcriptionally inactive phase, which becomes demethylated and transcriptionally active with 5-aza causing the cells to differentiate into myogenic cells.To investigate the ability of MNCs for improving damaged heart function and explore the optimal delivery period, 30 Japanese white rabbits at 5 months of age wererandomly divived 6 groups: Sham-operated group, Non-treated group, MNCs-treated group at 3 days after myocardial infarction(Ml), MNCs-treated group at 1 weeks after MI, MNCs-treated group at 4 weeks after MI, and MNCs-treated group at 8 weeks after MI. MNCs were isolated from rabbits' tibias on the second day after coronary ligation. Labeled with bromodeoxyuride (BrdU), the cells were auto-transplanted into bordering zone of the infarct. The animals were killed at 2 months after transplantation. The left ventricular function, infarct size, and capillary density were measured and the hearts were analyzed for the differentiation of the implanted bone marrow mononuclear cells. The main results in our research showed that (1) autologous implantation of BM-MNCs can express systolic proteins specific for cardiomyocytes, such as cardiac specific troponin T; (2) BM-MNCs were incorporated into capillary and arteriolar vessel walls. MNCs induced angiogenis as well. Although capillary density were significantly greater in all the MNCs-treated groups than the Non-treated group, the difference among the MNCs-treated groups did not reach statistical significance; (3) implantation of BM-MNCs can induce myocardial repair, improved left ventricular function, as well as attenuate LV transmural infracted area expansion and free wall thinning; Measurements of hemodynamics and anatomies of the MNCs-treated hearts showed that LV performance was sig...
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