Effects Of Oxygen Free Radicals And Calcium On Apoptosis Of Myocardium And The Influences Of Taurine,Amiodarone,Potassium Channel Opener,etc

Research Background and AimApoptosis is a special kind of cell death. It is an active cell "suicide" process which is regulated by the genes and distinct from cell necrosis. Necrosis has long been recognized as the unique form of cell death in myocardium. But recent researches showed that apoptosis contributes to the pathological process of myocardial ischemia-reperfusion injury,which is co-regulated by the apoptosis-inhibiting gene Bcl-2 and the apoptotsis-promoting genes bax, fas,bad and p53 etc.Apoptosis is a new issue in the field of ischemic heart disease.But at present,there are still different opinions about whether ischemia/hypoxia alone can induce myocardial apoptosis.The mechanisms of myocardial ischemia-reperfusion injury have also not been clarified.In recent years,effects of oxygen free radicals and calcium overload on myocardial ischemia-reperfusion injury have been highly regarded.But it is still not clear what roles they play in occurrence of apoptosis during myocardial ischemia-reperfusion injury.Among the various methods to protect myocardial ischemia-reperfusion injury, ischemic preconditioning(IPC) has been paid much attention to.But its correlation with myocardial apoptosis is not fully studied,especially the role of KATP channel. In recent years,antagonisms of taurine and amiodarone on oxygen free radicals and calcium overload have gained much attention.But no report was found about their effects on myocardial apoptosis.Aspirin is widely used to prevent and treat ischemic heart diseases,wheras it has an adverse effect of inhibiting the synthesis of myocardial prostacyclin. Whether aspirin can thus increase occurrence of myocardial apoptosis or not? Clarifying this issue is very important to clinical practice.This thesis will make a systematic study on these questions to discuss the roles which apoptosis plays during myocardial ischemia-reperfusion injury and oxidative stress,and to discuss the possibility and value of inhibiting myocardial apoptosis byanti-free radicals and anti-calcium overload medication or methods. MethodsNeonatal SD rat cardiomyocytes were isolated and cultured in vitro,with which to establish models of myocardial hypoxia-reoxygenation, simulated ischemia-reperfusion and oxidative stress induced by hydrogen peroxide. In vivo models of myocardial ischemia-reperiusion (I-R) and ischemic preconditioning (IPC) were established with adult SD rats. Taurine, amiodarone,pinacidil, aspirin etc were adopted as intervening factors respectively and different groups were randomized. Terminal deoxynucieotidyl transferase-mediated dUTP nick end labeling (TUNEL), propidium iodide(PI) staining and double-staining with flow cytometry were adopted to detect the apoptotic rates. Transmission electron microscope(TEM) and fluoromicroscope were used for morphology. Agarose gel electroghoresis was made for DNA ladder.Expression of apoptosis-related genes including bcl-2,bax,bad and p53 were detected by immunohistochemistry. These were used as apoptosis indicators.The extent of myocardial necrosis was reflected by detecting range of myocardial infarction, release of myocardial enzymes and the cell livability. The oxidative level was reflected by examining activity of superoxide dismutase(SOD) and quantity of malondialdehyde(MDA) in myocardium. Intracellular calcium concentration was detected by laser scanning confocal microsopy.Data in the results was statistically analized with SPLM software. Results and Conclusions1.Detection of apoptosis needs to combine several methods together,among which Annexin V-FITC stain can recognize apoptotic cells at an early phase(from the second to third hour) and can distinguish apoptosis from necrosis with high specificity and sensitivity,thus being an ideal method to detect apoptosis at present.2. After two hours of hypoxia followed by six hours of reoxygenation, as well as features of necrosis,the cultured neonatal rat cardiomyocytes showed a positive TUNEL staining,typical apoptotic cells under TEM,a clear sub-diploid peak with...