| Objective:It has become a hot area that to research biological pacemaker by transfecting the target gene into stem cells recently. Relative to the other way, this way are becoming mature, but their main weakness is the lack of pacing performance. In order to screen better construction methods, this study compared two commonly used seed cells and compared three commonly used transient transfection ways. We transfected HCN2and HCN4into stem cells with different proportions and used patch-clamp technique to explore the similarities and differences of stem cell electrophysiological properties between single-gene transfection and multi-gene transfection. And the final object is to provide theoretical and experimental basis for the future to build a more rational and efficient biological pacemaker.Methods:1. Compare the morphological features, proliferative capacity, differentiation capacity, transfection efficiency, Level of apoptosis between ASCs and BMSCs, and then screen the better one of them as a seed cell for biological pacemaker.2. Compare the transfection efficiency, operability, repeatability and the level of damage to cell growth among electroporation, cationic liposome method, and the calcium phosphate method, and then screen the best one of them to transfect the HCN channels to stem cells.3. Transfect HCN2and HCN4into stem cells with different proportions and used patch-clamp technique to explore the similarities and differences of stem cell electrophysiological properties between single-gene transfection and multi-gene transfection.Results:1. There was no significant difference between ASCs and BMSCs of dog. All of them showed the typical morpholgy of fibroblast cell. The proliferative capacity of ASCs was better than BMSCs. The fifteenth generation of cultured ASCs cells still maitained a high level proliferative capacity. The adipogenic differentiation ability of ASCs was better than BMSCs and both osteogenic differentiation capacity of them was no significant difference. Transfection efficiency the fifteenth generation of cultured ASCs cells was better than BMSCs and the level of damage to cell growth was lower than BMSCs.2. After transfected HCN4gene into ASCs, stem cells showing a green fluorescence under a inverted microscope. This prompted the green fluorescent protein was successfully expressed in stem cells. After transfection48h, we used immunohistochemistry to detect transient transfection efficiency, and results of calcium phosphate method, liposome method and electroporation were (8.62±1.13)%,(18.30±2.50)%and (25.32±5.40)%respectively. The transfection efficiency of electroporation is significantly higher than others methods (p<0.05). We compared the mortality rate of stem cells24h,72h and1week after transfection. The results showed that liposome method was slightly better than other two methods and the difference between calcium phosphate method and electroporation was not obvious.3. The channel currents recorded from stem cells after transfection were inhibited by Ivabradine (10μM/L) and the reduction of current amplitude was more obvious when CsCl (5mmol/L) was droped into the perfusion slot. These phenomenons prompted that the current we recorded was If. The results showed that the activation speed of HCN2channel was faster than HCN4. The current amplitude of stem cells which transfected multi-genes were higher than single gene transfecting cells. Activation time constant of multi-genes transfect groups were shorter than HCN2gene transfect group and HCN4gene transfect group, rather than between them.Conclusion:1. Dog’s ASCs has a stronger in vitro proliferative capacity than BMSCs, which is the basic capacity of the excellent seed cells. ASCs has a stronger cardiac differentiation capacity than BMSCs, therefore, it is more suitable for cardiac tissue engineering research. ASCs has higher transfection efficiency of plasmid pcDNA3.0-EGFP-hHCN4 than BMSCs and the rate of apoptosis is lower than BMSCs. As a results, ASCs is more suitable as a seed cell for building a biological pacemaker than BMSCs.2. Among three transiently transfection methods, electroporation has the highest transfection efficiency. Although transfection has high cell death rate after trasfection relative to the others methods, the difference between tham is not obvious. Moreover, it is foreseeable that we can reduce the cell death rate by optimizing the experiment parameters. Therefore, for the ASCs transfection of HCN, electroporation is more suitble.3. Electricity physiological characteristics of the channel currents after transfection is in accordance with HCN channels. The activation speed of HCN2channel was faster than HCN4. Activation time constant of multi-genes transfect groups were shorter than HCN2gene transfect group and HCN4gene transfect group, rather than between them. This suggest that maybe some synergies effects had been generated in the process of co-expression of HCN2and HCN4or there have some adjustment mechanism between the HCN2and HCN4. |
PDF Full Text Request