| Along with the development of technology and economy, the problem of population aging has been more conspicuous, that has led to the increase of the morbidity of the heart diseases which are related with age. Ischemic heart disease (IHD) and heart failure have been the leading threat to human health in all of the world, regardless the developed countries in Euro-American or our country. IHD is characterized by the reduce, necrosis and apoptosis of cadiocyte, what are caused by the imbalance of the supply and demand of oxygen. The main factors of cardiac function are the myocardial fibrosis and the formation of myocardial scar, which are also the Pathologic basal of serious heart failure, even death. In recent years, with the development of the molecular biology and the cell biotechnology technology and the further research of marrow stroma cells (MSCs), to substitute necrosis myocardial cells and to enhance heart function, by transplanting MSCs into the damaged cardiac tissue will be a new treatment strategy for the IHD. The research of the transplantation of stem cells to treat the myocardial infarction (MI) still place in a beginning stage, and those, the further plasticity research and application in the clinical treatment of IHD of MSCs, provide experimental and applied references for the potency of the MSCs clinical application. But there are a series of basal and applied problems to be illuminated before the large-scale clinical application of MSCs.Objective: to understand the biological property of MSCs, to observe the transplantion and proliferation of MSCs in different stages of MI and with different doses of transplanted cells and the amelioration of cardiac function in the model, and to investigate the best time of the cell transplantation and doses of the cells after MI, by establishing a vitro cultivation system of MSCs in advanced-age rabbits, those provide prophase experimental basis for the treatment of the diseases correlating to cadiocyte necrosis by cells transplantatio.Materials and Methods: The first fraction: sixteen New Zealand big-ears albino rabbits, aging 12-46 months (a 36-months rabbit is the advanced age rabbit, equaling a person 60 years old), were selected. Then we sampled their bone marrow blood, separated rMSCs by the Percoll (1.073g/ml), resuspensed the separated cells by L-DMEMs containing 10% of the fetal calf serum, inoculated cells into 24-bore plates with 3-4×106/cm2, and identified MSCs by associated methods (including immunohistochemistry, flow cytometry, induce-differentiation reverse identification). At last we observed the effects upon rMSCs of: 1. the donor natural conditions; 2. the different inoculation density; 3. the different frequency of changing nutrient solution in the same inoculation density; 4. the different passage ratios; 5. the different serum density.The second fraction: according to above-mentioned results, we selected New Zealand big-ears albino rabbits, which were older than or equal to 36 months. Model of MI was made by anterior descending branch band. Then we observed the effects of the cell transplantation upon heart function in condition of: 1.the same cell-density but different time of myocardial infarction;2. the same time of myocardial infarction but different cell-density.Results: The first fraction: the relation between the rMSCs growth rate and: 1. the donors age: There were significant differences in P1day between the rabbits age>36 months and the ones age<36 months, that demonstrated the effect of age on the rMSCs growth rate. But the third generation of hMSCs abstracted from different-ages donators, which were all over 106order of magnitude, could execute self-repaire completely. 2. the different inoculation density: the P1 day of the low density inoculation group was the longest, about 35 days, which was significant different (P<0.05) with the medium density group and the high definition one; with the comparison of the high definition group, the P1 day of medium density one prolonged obviously, and the high definition met the requests of the experimental and clinical application, of which cell grows most fast. 3. the different inoculation frequency: the quantity of attached cells was the least in the group which nutrient solution was changed in the second day, and there were no significant differences (P<0.05) between 3 days (8-33 cells/well) group and 4 days group (36-54 cells/well). 4. the different passage ratios: the shortest P2-P3 time was in the group with passage ratio 1: 2, and the longest in the one with ratio 1: 4. 5. the different serum density: there were no significant differences (P>0.05) between the group with 8%-10%FCS and the one with 20%FCS by rank-sum test, that meaning 20% FCSs didn't accelerate the cells growth rate; the cell growth rate was obviously slower in the group without serum, and there were significant differences (P<0.05) compared with the groups with serum.The second faction: 1. Compared with the control group, in the condition of the same cell-density but the different time of myocardial infarction, all the indices of heart function raised in the group of 1 day, 1 week, 2 weeks, 3 weeks and 4 weeks, and the differences in the group of 1 week, 2 weeks and 3 weeks were the most significant (P<0.01), which were better than the first group (P<0.05), however without significant difference inter-comparison (P>0.05); there were no significant difference (P>0.05) between the last group and the control one. Although the transplantation time window was 1-21 days after MI, which was advantageous to the decrease of MI area and the amelioration of heart function, the best time was 2 weeks after MI. 2. The high inoculation density (4×106) was advantageous to heart function with the same time of myocardial infarction and the different cell-density. Conclusions: 1. MSCs of New Zealand big-ears albino rabbits could be separated effectively by the Percoll centrifugalization combined with selectivitive attached screening method. 2. There were negative correlations between the donor age and rMSCs grow rate, but the numbers of cells amplification in different groups all satisfied the demand of clinical applications. 3. In the same inoculation density, the best time for the first changing liquid is 3-4 days. 4. The suitable serum density for rMSCs growth was 8-10%, the increment serum concentration could not increase the growth speed, but increase cells differentiation. 5. The P1 day of the low density inoculation group was the longest, the high density met the requests of the experimental and clinical application, of which cell grows most fastest. 6. The shortest P2-P3 time was in the group with ratio 1: 2, and the longest in the one with 1: 4. 7. In the condition of the same cell-density but the different time of myocardial infarction, the transplantation time window was 1-21 days after MI, which was advantageous to the decrease of MI area and the amelioration of heart function, but the best time was 2 weeks after MI. 8. The high inoculation density (4×106) was advantageous to heart function with the same time of myocardial infarction and the different cell-density.In this study, we formulated the vitro abstraction, cultivation and transplantation in advanced-age rabbits for the first time, understood the biological property of MSCs, investigated the best time of the cell transplantation and doses of the cells after MI initially, at last discovered the suitable number and the best time of MSCs transplantation after MI. Nowadays, the problem of population aging has been more conspicuous, and IHD have been the leading threat to human health. That, how transplanting the MSCs as stem cells into the microenvironment of impaired myocardium, to substitute the necrosis cadiocyte and the cicatricial tissue, with purpose of increasing competent myocardium and enhancing heart function, will be the theoretical and practical foundation of the treatment of IHD. |
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