| Backgrouds:Recent advances in molecular and cell biology and in tissue engineering technologies have paved the way to develope a new and exciting field in biological pacemaker. Many researches used the method by carrying different genes to seed cells to build biological pacemaker. They transplanted seed cells transferred pacemaker channel into heart model and made it showing ectopic pacing activities. These studies took the first step toward successful constructing biological pacemaker. Thus, choose good seed cells and an ideal pacing gene are particularly important. Because bone marrow stem cells are the first adult stem cells to be found in the world, many scholars choose it as seed cells to build biological pacemakers. More focused on the choice of pacing related genes. The most popular genes are HCN2. In recent decades, with the in-depth study on sinus node, HCN4 become more and more attractive. So far, there was no large breakthrough about building biological pacemaker. The reason is probably that there are not ideal seed cells and genetic combinations. In other words, seed cells may be the most important factor in building biological pacemakers. In recent years, Adipose tissue derived mesenchymal stem cells (ADSCs) are more and more concerned. People speculate that it will become a kind of better seed cells. But, whether each species ADSCs are suitable to serve as the seed cells? Therefore, further study about biological characteristics of ADSCs in different genus and became important. In addition, we compared the biological features between ADSCs and BMSCs, especially in constructing biological pacemaker. At last we could find the most ideal biological pacemakers seeding cells.Objective:The aim of this study was to analyze the differences in proliferation, phenotype and differentiate capacity of ADSCs from three different species respectively. A comparative study of cultured rat, canine and human ADSCs were carried out, and the main morphological parameters, proliferative activity, expression of surface markers, multi-differentiate capacity, gene transfection efficiency and apoptosis rate were characterized. Then, we compared BMSCs and ADSCs cultured under identical conditions with respect to culture characteristics, proliferation capacity, cell cycle, multi-differentiation potential, cardiomyogenic differentiation capacity, gene transfection efficiency, apoptosis rate, gene and protein expression of HCN2 and HCN4 in BMSCs and ADSCs in vitro at different passages. We also studied the relationship between HCN2 and HCN4 mRNA level and time index after gene transfection. So we could find out which are suitable seed cells in biological pacemakers study.Methods:Adipose-derived stem cells (ADSCs) were isolated with standard methods. To further study the proliferation and multi-differentiation potentials of ADSCs, their growth curves were achieved with cck-8. Surface protein expression was analyzed by flow cytometry and the multi-lineage potential of ADSCs were testified by differentiating cells with adipogenic and osteogenic inducers. Alkaline phosphatase (ALP) staining and Oil-red O staining were carried out 28 days later. After transfecting the three kinds of ADSCs with plasmid of pacemaker gene pcDNA3.0-EGFP-hHCN4, we compared the gene transfection efficiency and apoptosis rate. Then we extensively evaluated the differences between rat BMSCs and ADSCs in culture characteristics, proliferation capacity and cardiomyogenic differentiation capacity. The expression of cardiac pacing-related genes, including hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) and HCN4 were detected by realtime-PCR. Western blot was used to detect the corresponding protein expression. The cell apoptosis rate and gene transfection efficiency were detected by flow cytometry after transfecting plasmid. At last, we detected the expression of HCN4 mRNA 2 weeks after transfecting plasmid.Results: 1. The results showed that no significant difference was observed for the morphology of ADSCs under light microscope. We examined the expression of CD29, CD44, CD73 and CD34 of three species ADSCs. CD29, CD44 and CD73 were positive in expression, while CD34 was negative. The expression level of CD29 and CD44 had no significant differences among three ADSCs, but the percentage of CD73 was detected with great variability. CD73 was expressed highly in human ADSCs, but the expressions in canine's ADSCs were much lower. In this study, the cells were able to differentiate into adipocytes and osteoblasts in vitro, strongly suggesting that the cells we obtained were ADSCs.2. The proliferation capacity was similar in all animals. Rat ADSCs showed higher adipogenic and cardiomyogenic ability and dog showed higher osteogenic ability. There were a 3700bp balteum and a 5400bp balteum in pcDNA3.0 hHCN4 after cutting by EcoRI and XBaI, which was identical with the size of the objective gene. Many cells gave out green fluorescent after treated by pcDNA3.0-EGFP-hHCN4, the carrying efficiency between 30-40%. The apoptosis rate of human ADSCs was much higher than the other two species ADSCs.3. CCK-8 test found group showed that ADSCs possess high self-propagating potential. Cell circle stage was measured by flow cytometry assays. The ratio of S phase of rat ADSCs was higher than BMSCs. The capacity of ADSCs differentiated into cardiomyocytes was significantly higher than that of BMSC; meanwhile, ADSCs retained the high cardiac differentiation capacity up to passage 10. However, the cardiomyogenic differentiation capacity of BMSCs decreased significantly at passage 10. We found that the gene transfection efficiency declined in BMSCs at passage 10 and the apoptosis rate of BMSCs was higher than ADSCs at the same passage. We also found the protein expression levels of HCN2 and HCN4 were higher in the ADSCs than BMSCs at the same passage. Meanwhile, the expression levels of these proteins decreased in BMSCs at passage 10. In all groups, we detected the expression of HCN4 mRNA after treating by pcDNA3.0-EGFP-hHCN4. The transfer efficiency of ADSCs and BMSCs at passage 5 was significantly higher than ADSCs and BMSCs at passage 10. Two weeks after transferring with pcDNA3.0-EGFP-hHCN4, the hHCN4 mRNA expression decreased in ADSCs and BMSCs at passage 10, but the decreasing level was higher in BMSCs.Conclusion:We could obtain ADSCs from human, canine and rat by standard method. ADSCs have strong multiplex differentiation capacity. Because rat ADSCs have higher cardiomyogenic differentiation ability, but the apoptosis rate was lower. Therefore, ADSCs maybe good kind of seeding cells. After further compared with BMSCs, ADSCs have higher self-propagating potential, gene transfection efficiency, HCN4 mRNA expression level and lower apoptosis rate. We concluded that rat ADSCs were more suitable for biological pacemakers study as seeding cells. |
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