Influence On The Proliferation Of Primary Cardiomyocytes By Transfection Cyclin A2 Gene In Vitro

BackgroundCurrently, the clinical treatment of cardiovascular malignant events mainly depends on taking drug to control and surgical to intervention. In view of the treatment and long-term benefits, the mortality rate of myocardial infarction just got a temporary decrease, meanwhile, the mortality risk of heart failure greatly exacerbated in virtually. In recent years, stem cells replacement therapy as the leading position of the experimental research has been widely developed, but impacted by the source of cells, transplantation risks and other objective factors, while limited by ethical constraints and other subjective factors, which yet to be implemented in clinical applications at a large scale. As known to all, the proliferation ability of mature cardiomyocytes was very weak, so in the face of sharply and severely hypoxic-ischemic injury a large number of myocardial cells came to be necrosis, resulting in heart function damage and even heart failure. If mature cardiomyocytes can be induced to continue devision and proliferation, effective proliferation of the cardiac regeneration is expected to be completely overcome the problem. Nevertheless, there were still many issues about myocardial cells regeneration remain to be resolved.ObjectiveTo investigate the influence on proliferation of primary culture of neonatal rat cardiomyocytes transfected by Cyclin A2 gene in vitro, provide cytological evidence for cardiac regeneration.Methods1. Cardiomyocytes of neonatal rat heart were digested repeatedly by collagenase type I and trypsin, combined with the differential attachment technique and BrdU in order to reduce the fibroblast content. When the myocardial cells were placed into constant temperature incubator to incubate, we could monitor cardiomyocytes with well morphological change and spontaneous beating by live cell imaging system.2. In this study, the primary cultured cardiomyocytes were divided into three groups as follows. (1) Ad-Cyclin A2-GFP Group, cells were transduced with adenovirus expressing Cyclin A2 and GFP. (2) Ad-Null-GFP group, cells were transduced with andenovirus expressing only GFP. (3) Control group, Non-transfection cells were used as negative control. The GFP was employed to determine transduction efficiency.3. The GFP tracing technique and statistical analysis were used to estimate the recombinant adenovirus transfection efficiency. Compared Ad-Cyclin A2-GFP group with Ad-Null-GFP group, we would see if there was difference intransfection efficiency between above two groups.4. The expression of Cyclin A2, phosphorylated histone H3 (H3P), cardiac troponin-T (cTnT) were detected by immunofluorescence at day 3-5 after cardiomyocytes were transduced with andenovirus.Results1. The vast majority of cardiocytes had grown adhering to the dish since 8-12 h, and we would see some single cell beating at 13-44 per minute. After 3-5 days, those cardiomyocytes converaged together, formed monolayer cells, and beated spontaneously and rhythmically at 46-182 per minute.2. Analysis of eGFP expression showed that the transduction efficiency of Ad-Cyclin A2-GFP group was (97±0.74)%, and the result of Ad-Null-GFP group nearly reached 100%. Thus it can be seen that there is no obviously difference between two above-mentioned groups. We adopted live cell imaging system to monitor the transferred cardiac myocytes still beating rhythmically and synchronously with green fluorescence.3. The consequence of immune-fluorescent staining presented Ad-Null-GFP group was similar to control group. As specific marker protein for myocardial cells, cTnT was mainly found in the cytoplasm in GFP-expressing and untransduced cells, suggesting that adenovirus/GFP has no toxicity in the cultured cardiomyocytes. Immuno staining results indicated that the percentage of isolated cTnT positive cells is (95±0.62)%. CyclinA2 was expressed mainly in the nucleus. H3P was also localized in the nucleus as one kind of nuclear protein, phosphorylated during mitosis phase.4. In the Cyclin A2-overexpressing cells, the percentage of H3P positive cells was significantly increased compared to those untransduced or only GFP-expressing cells (p<0.05). The experimental group showed a large number of multinucleated cells, in which the dual-core cell-based, followed by three nuclear cells.Conclusions1. It can be effectively and quickly cultured a great quantity cardiomyocytes with well morphology and spontaneous beating by this method, including collagenase type I trypsin, and differential paces of sticking to wall technique.2. Adenoviral mediated target gene transferred to primary cardiomyocytes with high transfection efficiency. The average transfection rate was more than 96%.3. H3P positive rate was available to stand for the chromosome mitotic index, so it is a strong evidence for myocyte proliferation.4. Cyclin A2-overexpression in primary cardiomyocytes promoted cardiomyocytes proliferation to form a dual-core. Sometimes we could also see a small number of multi-nucleated myocardial cells.