Quantitation Of DNA Breaks And Its Application In Research Of Apoptosis Analysis

Objectives:(1) To establish a assay for DNA breaks quantitation;(2) To explore the relationship between apoptosis and spontaneous hypertension, and the effect of angiotensin converting enzyme(ACE) inhibitor on apoptosis;(3) To investigate the role of purinergic P2z receptor for apoptosis of human leukemic lymphocytes mediated by extracellular adenosine triphosphate (ATP);(4) To investigate the role of bivalent cations and choline in ATP-induced apoptosis of human leukemic lymphocytes via P2z purinoceptor.Methods:(1) Based on saturation labeling 3'-ends of DNA fragments withα³²P dCTP in the presence of 2',3'-dideoxy-cytidine-5'-triphosphate(ddCTP) by terminal deoxy -nucleotidyl transferase(TdT). The saturation labeling 3'-ends of DNA fragments was performed by adding different concentration ofα³²P dCTP to a DNA sample, from which a maximal labeling (Lmax) and a kinetic parameter (Km) of the TdT reaction are calculated. The saturation labeling gives true quantitaion that make it possible to accurately compare quantities of DNA fragments among different samples. In the same time, we compare the accuracy of this method and other analytic method of apoptosis,such as transmission electron microscopy, DNA gel electrophoresis, flow cytometric analysis and TUNEL.(2) Male spontaneously hypertensive rats(SHR) and normotensive control rats(WKY) were used, meanwhile, the treatment of SHR with ramipril, an inhibitor of angiotensin converting enzyme inhibitor (ACEI) was administered orally (1mg/kg/d) to SHR from 3 to 10 or from 5 to 10weeks of age. Apoptosis in cardiomyocytes of SHR was quantified by a maximal labeling (Lmax) method and the characteristic features of apoptosis were identified by transmission electron microscopy (TEM), in situ labeling of DNA strand breaks with terminal deoxynucleotidyl transferase mediated dUTP and labeling (TUNEL)...