大鼠心肌延迟缺血再灌注损伤模型的建立及其心功能和心肌细胞凋亡情况观察  

Establishment of rat models with delayed myocardial ischemia-reperfusion injury and apoptosis of myo⁃cardial cells

作　　者：唐咏文 崔同涛 刘勇[1] 王敏 郭涛 刘津 谭宁[1] TANG Yongwen;CUI Tongtao;LIU Yong;WANG Min;GUO Tao;LIU Jin(Guangdong Provincial People's Hospital(Guangdong Academy of Medical Sciences),Guangzhou 510080,China)

机构地区：[1]广东省人民医院(广东省医学科学院),广州510080 [2]广州医科大学附属第一医院

出　　处：《山东医药》2021年第5期18-21,25,共5页Shandong Medical Journal

基　　金：国家自然科学基金项目(81270286)。

摘　　要：目的构建大鼠心肌延迟缺血再灌注损伤模型,并通过观察其心肌细胞凋亡情况及心功能变化加以验证。方法取SD大鼠42只,结扎心脏前降支血管制作心肌缺血模型,分别于结扎后1、4、6、12、24 h后打开线结观察5 min内再灌注情况以确定及时和延迟再灌注时间。将SD大鼠分为假手术组、及时再灌组和延迟再灌组,及时再灌组和延迟再灌组心肌缺血后分别按确定的时间予以再灌注,假手术组仅穿线不结扎。再灌注24 h,采用TTC/Evens Blue双染色法测算及时和延迟再灌组的梗死区、危险区面积、缺血区大小,改进TUNEL法检测三组缺血危险区心肌细胞凋亡情况,采用Western blotting法检测心肌细胞凋亡相关蛋白Bax、Bcl-2及激活态Caspase-3(Cleaved Caspase-3);再灌注72 h,采用动物超声仪检查左心室形态及心功能指标左心室收缩末直径(LVDs)、左心室舒张末直径(LVDd)、左心室短轴缩短率(FS)、左心室收缩末容积(LVDsV)、左心室舒张末容积(LVDdV)、左心室射血分数(LVEF)、每搏射血量(SS)和心输出量(CO)。结果缺血12、24 h后均无法成功再灌注,缺血6 h后10只中仅2只成功再灌注,缺血1、4 h后均成功再灌注,将缺血1 h和4 h分别作为及时和缺血再灌注时间。与及时再灌组比较,延迟再灌组坏死区、缺血区均增大(P均<0.05),危险区差异无统计学意义。及时再灌组和延迟再灌组的心肌细胞凋亡率高于假手术组,且延迟再灌组高于及时再灌组(P均<0.01)。与假手术组比较,及时再灌组和延迟再灌组Bax、Cleaved Caspase-3表达均升高(P均<0.05或<0.01),Bcl-2蛋白无明显变化。与假手术组比较,延迟再灌组和及时再灌组FS、SS、LVEF、CO均下降(P均<0.01),且延迟再灌组LVDsV和LVEF均低于及时再灌组(P均<0.01)。结论SD大鼠心肌缺血4 h后可有效再灌注,并可导致明显的心肌细胞凋亡及左心室收缩功能下降,该模型可更好地模拟临床心肌延迟缺血Objective The models of delayed myocardial ischemia-reperfusion(I/R)injury in rats were constructed and verified by observing the apoptosis of myocardial cells and changes in cardiac function.Methods Forty-two SD rats were selected and suffered to the myocardial reperfusion after the anterior descending artery ligation for 1,4,6,12,and 24 h.The optimal ischemia time was selected with successful reperfusion appearance of anterior wall color and ST segment elevation recovery within 5 minutes.SD rats were divided into three groups including the sham operation group,timely re⁃perfusion group,and delayed reperfusion group.Timely and delayed reperfusion cut-off time depended on the successful myocardial reperfusion appearance.Rats in the sham operation group were not ligated.The infarct area(IA),risk area(AAR)and total ischemic area(IA+AAR)after reperfusion were measured and calculated by Evens Blue/TTC stainingat 24 h after reperfusion.Modified TUNEL assay was applied to detect the apoptosis of cardiomyocytes in the ischemic risk ar⁃ea of the three groups.The expression of apoptotic proteins Bax,Bcl-2 and cleaved Caspase-3 were detected by Western blotting at 24 h after reperfusion.The morphology and function indexes of the left ventricular end-systolic diameter(LVD),left ventricular end-diastolic diameter(LVDd),and left ventricular short-axis shortening rate(FS),left ventricular end-systolic volume(LVDsV),left ventricular end-diastolic volume(LVDdV),left ventricular ejection fraction(LVEF),ejection fraction per stroke(SS)and cardiac output(CO)were measured by the animal ultrasound machine at 72 h after reperfusion.Results Reperfusion was not achieved after 24 or 12 h of permanent ischemia.Only 2 of 10 rats were successfully perfused after 6 h of ischemia.One-hour and four-hour continuous ischemia were regarded as timely and delayed ischemia-reperfusion time,respectively.Compared with the timely reperfusion group,the necrotic area and isch⁃emic area of the delayed reperfusion group increased(both P<0.05),and the

关 键 词：缺血再灌注 细胞凋亡 左心室功能 BAX Bcl-2 CASPASE-3 大鼠 

分 类 号：R541.4[医药卫生—心血管疾病]