| Coronary atherosclerotic heart disease may play a major role in the world. It is one of the most common factors to cause ischemic heart disease. Ischemia is accompanied by changes in the Oxygen state and conditions. Ca2+is respond to the ischemic injury of the heart. Ca2 + has been postulated to play an important role in modulating coronary atherosclerotic heart disease. Furthermore, we often applicated to cure coronary atherosclerotic heart diease for extracellular Ca2 + influx which this therapy was lack to choice and has side effect. It was new outbreak that therapy measures of cardiac hypoxia were mediated from intraceUular Ca2+ release. We know that there was the report about pharmacological study of dehydeocorydaline cure to coronary atherosclerotic heart disease in 1978. But almost few article elucidated in the cardiovascular mechanism, particularly the mechanism of molecule biology is unknown in the world. In order to build and improve the method of a stable isolation and culture of neonatal rat myocytes, the ratio of the dissociation trypsin blue -negative cells and beat cells were increased. In order to study molecular mechanisms of dehydeocorydaline underlying calcium handling may protect cultured cardiomyocytes from injury induced by hypoxia. We studied the effect of Dehydeocorydaline and Verapamil( Ver) on intracellular free calcium concentration of myocardial cell ([ Ca2+ ];) under hy-poxic condition.MethodsBuild of isolated and cultured method of neonatal rat myocytes: The techniques used in our new method were based on Simpsons, and mice were killedby cervical dislocation. The heart was excised, arrested in ice - cold buffer, and sheared into 3 -5 mm partition. Neonatal rat cardiomyocytes were isolated with trypsin. The cardiomyocytes quality was evaluated with morphology and trypan blue dye. States of myocytes growing in culture were observed with optical microscope and electron microscope photography. The model of original cultured cardiac myocytes of neonatal rat was established.Methods of the Influence of dehydeocorydaline intracellular free calcium concentration during hypoxia in myocardial cell of guinea - pigs: We adopted self - control study and connected guinea - pig heart Langendorff instillation. Retrograde perfusion was maintained at 37C by using water -jacketing and e-quilibrated with 95% O2 and 5% C02( pH 7.4). The myocardial cells were isolated by collagenase ( Tyoe I, sigma) and marked by fluorescence ratio imaging. The suspension of myocardial cells was assigned to six groups: DHC, Ver, and control were each two. Each three groups equilibrated with 99% N2 gas in the hypoxia condition (PO2:7.7 -13.3kpa) . And was exposed to normoxia before determination of [ Ca2+ ];.Methods of the mRNA amounts and protein expression of these calcium handling genes: According to TRIzol Reagent kit. The mRNA amounts of these calcium handling genes including sarcoplasmic reticular ( SR) Ca2+ - ATPase, L - type calcium channel, ryanodine receptor, calsequestrin and phospholsmban were measured by reverse transcription -polymerase chain reaction (RT -PCR) and normalized to the mRNA levels of actin. The PCR program used was the following: 94% strand denaturation for 5minute, followed by a 3 step cycling program of 951 for 15seconds, annealing at 56C for 30 seconds, and extension at 72C for 30 seconds for a total of 35cycles. The last cycle was followed by a 3 minute extension at 72C. The products were run on PAGE gel electrophoresis. We used method of RT - PCR to examine the level of transcription of calcium handling genes in cultured cardiomyocytes to investigate the protective effect of dehydeocorydaline.Methods of the mRNA amounts and protein expression of these calcium handling genes: Samples load on to single - dimension SDS - PAGE. For immu-noblotting, proteins were transferred to nitrocellulose membranes using blottingtechniques. Membranes were blocked for lhour in 5%BSA - TTBS probed with these specific calcium handling genes antibody for overnight at 4C. And then...
|
PDF Full Text Request