Experimental Study Of The Effect Of Bcl-2 Gene On Ischemia Reperfusion Injury And Apoptosis Of Cardiomyocytes

Myocardial ischemia will occur when the coronary blood supply cann t meet the needs of the myocardial metablism. Once the ischemia occuerred, it will lead to not only the hypoxia and metablic disturbance, but also the accumulation of the toxic metabolic products which can cause the ischemic injuries and even the death of the myocardium. Early reperfusion is important to the survival of the myocardium, but the active oxygen produced by it will deteriorate the injury to the Cardiomyocytes. For a long time , the cell death has been regarded to be the sole style of this myocardial cellular death. But more and more studies have showed the myocardial cellular apoptosis participated in the pathological course of the myocardial ischemia reperfusioninjury. The cellular apoptosis is a new problem in the study of the ischemia heart disease.Apoptosis is an active, manipulative and normal phenomenon ,which is regulated by a series of gene. The gene therapy can affect apoptosis by charging the gene which regulate the apoptosis. Bcl-2 family are anti-apoptosis molecules which can inhibit apoptosis caused by many kinds of stimulates.The purpose of this study is to investigate the relationship between apoptosis and ischemia reperfusion injury, to confirm that Bcl-2 gene has the effect of restrain apoptosis of myocardium, so as to provide the experimental date to support the new genethrapy of ischemia reperfusion.Using the myocardial ischemia reperfusion rat model, we studied the relationship between myocardial apoptosis and reperfusion injury with different duration of reperfusion after myocardial ischemia. The myocardial cell apoptosis was determined with terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) method. The results were as follow: Cardiomyocyte apoptosis varied with the duration of reperfusion. The apoptotic index(AI) were highest in rats with 45min of ischemia followed by 2 and 4 h of reperfusion(I 45 min R2 and 4h) ( 24.2?.9 0/o; 20.2?.2?o); The cardiomyocyte apoptosis was positively correlated to the level of CK( r=0.92, p<0.05) and was negatively correlated to ventricular function (r=- 0.81 , p<0.05) . The results showed that: The number of apoptotic cardiomyocytes varied with different duration of myocardial reperfusion. The change of apoptosis may be involved in the process of myocardial ischemia reperfusion injury.Using the cultured rat myocardium, we studied the effect of the transfected anti-apoptosis Bcl-2 gene on cell viability and apoptosis in vitro. The research methods are as follow: The retrovirus eukaryotic expression vector pDOR -SB containing human Bcl-2 gene was extracted and identified by agarose gel electrophoresis after being cut with restricted endonuclease EcoR I . The pDOR - SB were introduced into cardiomyocytes by liposome-mediated gene transfection method. The transfection was identified with Northen blot hybridization. The growth properties and tolerance to ischemia reperfusion injury of the cardiomyocytes were studied in Bcl-2 gene-transfected and non-transfected cells. The results were as follow: The Northen blot hybridization showed that Bcl-2 gene was transfected in the most of the pDOR -SB-transfected-cell and it could increase the cell viability and hypoxic tolerance of cardiomyocytes. The research showed: Transfected Bcl-2 gene into cardiacmyocytes can significantly enhance the survival of cardiomyocytes under ischemia reperfusion condition.The study showed that transfected Bcl-2 gene can prevent ischemia reperfusion injury. It provides valuable date for the novel treatment of ischemia reperfusion with transfected Bcl-2 gene.