【作者】 王进；

【导师】 王一彪；

【作者基本信息】 山东大学 ， 儿科， 2005， 硕士

【摘要】 目的:1.心肌缺氧/复氧损伤是多因素所致的复合性损伤,但其具体机制仍不清。本文旨在探讨氧自由基在乳鼠心肌细胞缺氧/复氧损伤中的作用。2.研究表明,钙离子超载参与了心肌缺氧/复氧损伤,本文通过观察细胞内钙离子浓度在乳鼠心肌细胞缺氧/复氧损伤中的变化,显示钙离子超载在心肌缺氧/复氧损伤中的作用。3.探讨还原型谷胱甘肽（GSH）作为一种自由基清除剂及钙离子拮抗剂,对缺氧/复氧培养乳鼠心肌细胞损伤的保护作用。方法:取出生1～3天的Wistar大鼠,剪取心尖部组织,将心肌剪成约0.5～1mm³大小的碎块,用0.25%胰酶消化4～5次,收集除第一次消化后的上清液,将收集到的上清液在4℃、500r/min的速度下离心10min,弃去上清液,用平衡盐溶液洗涤细胞。然后用DMEM培养基配制成细胞悬液。应用差速贴壁法分离纯化心肌细胞。将原代培养的乳鼠心室肌细胞分为正常对照组与还原型谷胱甘肽不同浓度处理组。将正常培养72小时后的心肌细胞换用缺氧缺糖DMEM培养基,培养板放入充有99.5%N₂的密闭容器中,37℃密闭培养2h。将缺氧的心肌细胞换用饱含95%O₂+5%CO₂的含糖DMEM培养基,正常条件下培养1小时,建立缺氧/复氧损伤模型。缺氧、复氧前均给GSH0、40、80、160mg/L浓度,缺氧2小时,复氧1小时,在缺氧前、缺氧2小时及复氧1小时检测培养液中超氧化物歧化酶更多还原

【Abstract】 Objective:1.Anoxia/reoxygenation injury of myocardial cells is injured by many factors,the concrete machanism is not very clear.This study is to investigate the effect of the oxygen free radical in myocardial cells in rats ofanoxia/reoxygenation.2. Some researches show that the over-load of cytosplic free Ca²⁺ has a hand in anoxia/reoxygenation injury of myocardium.This article through having observed the change of cytosplic free Ca²⁺ after anoxia and reoxygenation injury to indicate the function of over-load of cytosplic free Ca²⁺ in myocardial cells of anoxia/reoxygenation.3. Reduced Glutathione（GSH） is a substance which can eliminate the free radical and perhaps can be regarded as an calcium antagoni.This study is to find out the protective effect of GSH on anoxia/ reoxygenation injury of myocardial cells in rats.Methods: 1 to 3 days old rats were used. The ventrical tissue of the hearts were selected.The specimen was cutted into pieces of 0.5 to 1mm³.Digesting 4 to 5 times with 0.25% pancreatin.All the supernatant solution except the first digesting solution was clectted,that was centrifuged10 minutes at 4"C , 500 r/min, then the supernatant was discarded. The remain cells, after had washed with balanced salt solution, were mixed to suspension with DMEM culturd midium, and were purificated to keep the myocardial cells out of others. Primary cultured myocardial cells of rats were randomly allocated to 5 groupsxontrol group just used normal saline;reduced glutathione groups were respectively given 0,80,160mg/L medicament before anoxia（2h） or reoxygenation（lh）. Measuring the activity of superoxide dismutase（SOD） and the level of malondiadehyde（MDA） before anoxia, 2 hours after anoxia and 1 hour after reoxygenation on each group.While measuring the cytosolic free Ca2+on 1 hour after reoxygenati .Results: 1.After anoxia and reoxygenation, cardiomyocytes contracted, tending to get round in shaoe and its pseudopods decreased. Most cardiomyocytes were not beating.As the increasing of the dosage of GSH,the change of cells were improved.2. In single anoxia/ reoxygenation group（0mg/L）, the activity of SOD was decreased, while MDA was increased after anoxia or reoxygenation.Compared to 2 hours after anoxia .the changes of the activity of SOD and MDA were more markedly on 1 hour after reoxygenati.The modle of group successfully brings about the over-load of cytosolic free Ca2+.The content of cytosolic free Ca2+ be increasing on 1 hour after reoxygenation （P<0.01）.3. Giving reduced glutathione could significantly attenuate the reduced SOD activity and the elevated MDA, cytosolic free Ca2+ （P< 0.01）.The protective onSOD, MDA ,cytosolic free Ca2+ in a dose-dependent manner and 160mg/L on SOD , cytosolic free Ca2+ showed the greatest protective effect（?<0.01）, approaching to control group（P>0.05）.Conclusion: 1.In the mechanism of the injury to cardiomyocytes by anoxia/reoxygenation,the free radical is one of the most important factors.2.The over-load of cytosolic free Ca2+ take part in the injury to cardiomyocytes by anoxia/reoxygenation,they may play a negative synergismrole.3.Glutathione could protect the myocardial cell from the injury by anoxia/ reoxygenation.That may be related to the ability of glutathione against the oxidant injury and inhibit calcium overload. Glutathione perhaps can be regarded as an clciumantagonis.更多还原