| Part I: Study of hypoxia-induced Apoptosis in cultured cardiomyocytes of neonatal ratObjective: We developed a new model of cardiac ischaemia in-vitro that mimics distinct features of ischaemic injury. To investigate the temporal characteristics and the possible molecular mechanisms involved in hypoxia-induced apoptosis in cultured cardiomyocytes.Methods: SD neonatal rat hearts cells were cultured in stresses of hypoxia, acidosis and stagnant incubation medium for 12-72h. In this environment, the cultured cardiomyocytes from 2-3d newborn SD rats were randomly divided into 5 groups: control groups, ischaemic 12h group (I12h), ischaemic 24h group (I24h), ischaemic 48h group (L48h) and ischaemic 72h group (I72h)- The Po2 and pH of the medium were detected; the cellular CPK activity and ATP-ase activity were also detected. The cultured cardiomyocytes of each groups were taken for detecting apoptosis of cardiomyocytes by terminal deoxynucleotide transferase-mediated deoxyuridine-biotin nick end labeling (TUNEL), flow cytometry, DNA gel electrophoresis, Single cell gel elecrophoresis(SCGE) assay , microscopy and transmission electron microscopy (TEM).Results: In this model, the Po2 and pH of the medium gradually decreased during the ischaemic insult, with 72-h PO2 values being 15.3±1.15mmHg and pH 6.85±0.06 respectively. The model triggered severe cell injury, including morphological degeneration, CPK release, beating impairment and ATP depletion. As early as 12h after hypoxia insult in this model, TUNEL-positive cardiomyocytes were detected.The cultured neonatal rat heart cells apoptosis indexes (AI) at 12-hour after hypoxia insult were significantly higher than those of the control group (P<0.01),After a 48-and 72-h ischaemic insult, the cultured neonatal rat heart cells apoptosis indexes (AI) was 24.4±2.14% and 45.4±4.22% respectively. TEM examination showed that apoptosis occurred in some cardiomyocytes as early as 24h after ischaemic insult, reached a peak at 48 to 72 hours.Flow cytometry of cultured neonatal rat heart cells after ischaemic insult at various time points showed that the percentage of apoptosis cells increased quickly. The percentages of apoptosis cells at 12 hour after ischaemic insult were significantly higher than those of the control group (P<0.01). After a 48-and 72-h ischaemic insult, the percentage of apoptosis cells was 35.6±2.14% and 48.72±6.40% respectively.Gel electrophoresis of DNA extracted from the cultured cardiomyocytes of each groups presented typical "ladder" break in two groups at 48-hour and 72-hour, DNA extracted from the cultured cardiomyocytes at 12-h and 24-h presented no "ladder" break. Single cell gel elecrophoresis(SCGE) assay showed that the DNA strands were damaged significantly after hypoxia for 12 hour in comparison with control group(p<0.01). Hypoxia increased the percentage of comet cells, tail length, tail moment in a time-dependent manner.Conclusions: These results indicate that this model of cardiac ischaemic can induce apoptosis of cultured neonatal rat heart cells, the efficiency of apoptosis is related to the time-course of cultured cardiomyocytes after ischaemic insult. These results demonstrate that neonatal cardiomyocytes subjected this simulated ischaemic model exhibit similarities to cardiac ischaemic: including the simultaneous appearance of necrosis, breakdown of cellular ATP, beating cessation and apoptosis. Apoptosis is an important form of cardiomyocytes death in cultured neonatal rat heart cells after ischaemic insult. SCGE was a fast and sensitive method to detect the DNA damages of the cardiomycytes induced by hypoxia.Part II: Experimental study on apoptosis-related-genes expression in apoptotic cardiomyocytes after ischaemic insult.Objective: To investigate the alterations of apoptosis related genes in culturedneonatal rat heart cells and the molecular mechanism of cardiomyocytes apoptosis following hypoxia or ischaemic insult.Methods: SD neonatal rat hearts cells were cultur... |
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