【作者】 王华军；

【导师】 陈晓辉；

【作者基本信息】 广州医学院 ， 心肺脑复苏， 2010， 硕士

【摘要】 背景和目的:心功能不全是导致心跳骤停患者复苏后死亡的重要原因之一。我们前期研究观察到促红细胞生成素(EPO)对大鼠心肺复苏后的心功能不全有改善作用,但对EPO改善心肺复苏后心功能不全的途径和机制尚不明确。为此本实验通过观察EPO干预对心肺复苏后心肌细胞凋亡率和细胞骨架蛋白的变化,探讨EPO改善心肺复苏后心功能不全的机制;通过体外心肌细胞缺氧/复氧损伤模型,观察EPO和蛋白激酶C(PKC)抑制剂白屈菜红碱干预后,体外心肌细胞凋亡及黄素蛋白自体荧光强度的变化,探讨EPO抗心肌细胞凋亡的机制,为临床预防和治疗复苏后心功能不全提供实验依据。方法:第一部分:取前期心肺复苏实验已制作的大鼠心肌石蜡组织块,分为正常组、CPR组和EPO组,每组8只。行Tunel染色观察各组心肌细胞凋亡率,SABC免疫组化法观察微管蛋白、结蛋白和肌动蛋白变化。第二部分:将SD大鼠乳鼠心肌细胞建立缺氧/复氧模型(H/R),并随机分为4组:①正常组;②缺氧/复氧组(缺氧10h,复氧4h);③EPO治疗组(缺氧/复氧同时,加EPO干预);④抑制剂组(缺氧/复氧同时,加EPO和PKC抑制剂联合干预)。流式细胞仪Annexin V FITC/PI法检测各组心肌细胞凋亡率,免疫荧光法观察各组细胞微管蛋白、肌动蛋白结构和荧光强度变化,共聚焦显微镜观察各组细胞黄素蛋白自体荧光变化,监测线粒体ATP敏感性钾通道(mitoKATP)开放情况。结果:第一部分:1. CPR组心肌细胞凋亡数(314.1个±30.7个)较正常组(165.2个±45.9个)明显升高(P<0.05)。EPO治疗组心肌细胞凋亡数为(242.1个±20.0个),与CPR组比较,凋亡细胞数明显减少,其差异有统计学意义(P<0.05)。2.正常组、CPR组、EPO治疗组大鼠肌动蛋白免疫组化平均光密度值分别(8.18±1.04、8.51±1.96、6.34±0.43)×10-2,组间两两比较差异均无统计学意义(P>0.05);微管蛋白免疫组化平均光密度值分别(8.87±1.85、7.35±0.26、9.70±0.13)×10-2,组间两两比较均无统计学差异(P>0.05);结蛋白免疫组化平均光密度值分别为(7.57±2.96、8.20±3.17、7.48±1.82)×10-2,组间两两比较均无明显统计学差异(P>0.05)。第二部分:1.缺氧/复氧组心肌细胞凋亡率较正常组明显增加{(42.5%±8.0%)对(13.9%±2.0%)},比较有统计学意义(P<0.05)。EPO处理降低了缺氧/复氧心肌细胞凋亡率(22.7%±5.0%),与缺氧/复氧组比较差异有统计学意义(P<0.05)。蛋白激酶C抑制剂阻断了EPO抗缺氧/复氧损伤后心肌细胞凋亡作用,其凋亡率(46.7%±17.0%)与EPO治疗组比较差异有统计学意义(P<0.05)。2.缺氧/复氧组心肌细胞肌动蛋白、微管蛋白荧光染色结构明显破坏,网状结构模糊不清,平均荧光强度值{(0.43±0.15)×10-2、(0.36±0.10)×10-2}较正常组{(5.0±1.6)×10-2、(8.2±3.6)×10-2}明显减弱,差异均有统计学意义(P<0.05),但与EPO治疗组{(1.8±0.20)×10-2、(1.7±0.36)×10-2}比较差异均无统计学意义(P>0.05)。3. EPO治疗组心肌细胞黄素蛋白自体荧光强度(2.20±0.11)×10-2较正常组、缺氧/复氧组、抑制剂组{(0.15±0.05)×10-2、(0.28±0.17)×10-2、(0.99±0.33)×10-2}均明显增强,分别比较差异均有显著统计学意义(P<0.05)。结论:1.减少心肌细胞凋亡是EPO改善窒息诱导大鼠心肺复苏后心功能不全的重要机制之一。2. EPO抗凋亡机制可能是通过磷酸化PKC,继而开放mitoKATP通道而起作用。3.缺血/再灌注(缺氧/复氧)对细胞骨架的破坏作用可能具有时效性,EPO对细胞骨架无保护作用。更多还原

【Abstract】 Objective:Cardiac dysfunction is a major reason of death after cardiopulmonary resuscitation in patient with cardiac arrest. We confirmed erythropoietin could improve rat cardiac dysfunction after cardiac arrest resuscitation in our preliminary study. In order to clarify the mechanism of erythropoietin improving myocardial function after cardiopulmonary resuscitation, the apoptosis index , cytoskeleton proteins changes of rats after resuscitation and cultured myocardial cells following hypoxia/reoxygenation injury were observed, and the flavoprotein fluorescence brightness change of cultured myocardial cells were detected.Methods:Part I:The rat heart specimens were collected form previous cardiopulmonary resuscitation model experiment and were divided into normal group, CPR group and the EPO group. Myocardial cell apoptosis index was detected by Tunel stained and tubulin, desmin and actin was observed by SABC immunohistochemistry.Part II:The cultured cardiomyocytes were extracted from neonatal rats and were divided into four groups: the normal group, the hypoxia/reoxygenation (H/R) group, the EPO group and the inhibitor (chelerythrine) group. The cells were stained with fluorescent dyes and apoptosis was assayed by flow cytometry. Immunofluorescence was used to observe tubulin and actin change of cultured cardiomyocytes. Flavoprotein fluorescence was measured by confocal microscope to indicate the mitoKATP channel activity. qResults:Part I:1. Compared with normal group, the apoptosis index of CPR group was significantly increased (314.1±30.7 vs 165.2±45.9, p<0.05). Compared with the CPR group , the apoptosis index of EPO group reduced significantly (242.1±20.0 vs 314.1±30.7, P <0.05).2. Optical density of actin immunohistochemistry in the normal, CPR and EPO groups were(8.18±1.04、8.51±1.96、6.34±0.43)×10-2 , respectively . There was no significant difference among these groups (P>0.05); Optical density of tubulin immunohistochemistry in normal, CPR and EPO groups were(8.87±1.85、7.35±0.26、9.70±0.13)×10-2 , respectively and there was no significant difference among these groups (P>0.05); Optical density of desmin immunohistochemistry in normal, CPR and EPO groups were(7.57±2.96、8.20±3.17、7.48±1.82)×10-2 and there was no significant difference among these groups (P>0.05).Part II:1. Compared with the normal group, the apoptosis rate of hypoxia / reoxygenation group significantly increased {(42.5%±8.0%) vs (13.9%±2.0%), P<0.05}. EPO alleviated the hypoxia/reoxygenation injury of cultured cardiomyocytes, the apoptotic rate (22.7%±5.0%) showed statistically significant difference compared with hypoxia/reoxygenation group (P <0.05). While the PKC inhibitor chelerythrine blocked the EPO myocardial protection during hypoxia/reoxygenation injury, the apoptosis rate(46.7%±17.0%) showed statistically significant difference compared with EPO treated group (P <0.05). 2. During hypoxia/reoxygenation injury, structures of the actin, tubulin protein were destroyed, network structure unclear in cultured myocardial cells. The fluorescence intensity was much weaker than normal group {(0.43±0.15)×10-2, (0.36±0.10)×10-2}, the difference was statistically significant (P <0.05), but there was no significant difference compared with the EPO treated group (P>0.05).3. Compared with the normal and hypoxia/reoxygenation group{(0.15±0.05)×10-2、(0.28±0.17)×10-2},the flavoprotein auto fluorescence intensity(2.20±0.11)×10-2 of EPO group was significantly increased. However, the brightness of cardiomyocytes flavoprotein fluorescence(0.99±0.33)×10-2 was observed reduction after adding the chelerythrine (P <0.05).Conclusions:1. Anti-apoptosis is one of the important mechanisms of EPO improving cardiac dysfunction after cardiopulmonary resuscitation in rats.2. EPO may produce the anti-apoptotic effect through the mitoKATP channel actived by PKC.3. The damage of cytoskeleton during ischemia / reperfusion may be time–dependent. EPO has no beneficial effect on the cytoskeleton.更多还原