促红细胞生成素对缺氧／复氧心肌骨架蛋白的作用

The effect of erythropoietin on myocardial cytoskeletal proteins after hypoxia/reoxygenation injury

目的从大体和细胞的角度，观察促红细胞生成素（EPO）对缺氧／复氧心肌细胞骨架蛋白：肌动蛋白（a-actinin protein）、微管蛋白（tubulin protein）和结蛋白（desmin protein）的影响，并初步探讨EPO对缺氧／复氧心肌骨架蛋白作用机制。方法经夹闭气管8min建立窒息性大鼠心脏骤停一心肺复苏动物模型。24只Sprague-Dawley大鼠随机（随机数字法）分为3组：健康对照（N）组、全心缺血／再灌注（I／R）组和EPO组。I／R组于窒息致心搏骤停后8min，予胸外按压和机械通气进行复苏，恢复自主循环（ROSC），监测复苏后心功能状态。EPO组CPR后于ROSC后3min注射EPO5000U／kg）。确定模型成功后观察120min，取心室肌，SABC免疫组化法观察微管蛋白、结蛋白和肌动蛋白变化。分离培养SD大鼠乳鼠心肌细胞，建立缺氧／复氧模型。培养心肌细胞随机分为4组：①正常（C）组；②缺氧／复氧（H／R）组（缺氧10h／复氧4h）；③EPO组（缺氧10h／复氧4h后予EPO10U／mL干预），免疫荧光法观察各组细胞微管蛋白、肌动蛋白结构及其荧光强度变化。结果动物实验中，N组、I／R组和EPO组结蛋白、微管蛋白和肌动蛋白的免疫组化平均光密度值（OD）组间比较差异均无统计学意义。细胞研究实验中，H／R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，平均荧光强度值较C组明显减弱，但与EPO组比较差异均无统计学意义。结论缺氧／复氧对心肌损伤作用具有时效性，EPO对缺氧／复氧损伤的心肌骨架蛋白无保护作用。

Objective To observe the impact of hypoxia/reoxygenation on myocardial cytoskeletal proteins （ot-actinin protein, tubulin protein, desmin protein） and to investigate EPO lessening the damage of myocardial cytoskeleton proteins in rats proved by culturing hypoxia/reoxygenation injured myocardial cells in presence of EPO. Methods The rat model of asphyxia-induced cardiac arrest was performed by turning-off the ventilator and clamping the endotracheal tube. After asphyxia for 8 minutes, CPR was carried out. A total of 24 rats were divided into normal group, ischemia/resuscitation （I/R） group and the EPO group （n = 8 ）. The model of myocardial dysfunction was determined 2 hours after restoration of spontaneous circulation （ROSC）. The rats of EPO group were given EPO 5000 U/kg after ROSC. The rat heart specimens were collected. Actinin, Tubulin and Desmin protein were observed by SABC immunohistochemistry. The cultured cardiomyocytes were taken from neonatal rats and were divided into three groups： the normal group, the hypoxia/reoxygenation （H/R） group （hypoxia 10 h/reoxygenate 4h）, the EPO group （hypoxia 10 h/reoxygenate 4 h, plus 10 U/mL EPO）. The changes of tubulin and actinin in cultured cardiomyocytes were observe by Immunofluoreseence. Results From immunohistochemistry, there were no significant difference in the optical density of aetinin, tubulin and desmin among the normal, I/Rand EPO groups. After H/R injury, the structures of the actinin, tubulin protein were destroyed, the network structure of both protein were unclear in cultured myocardial cells. The grades of fluorescence intensity of actinin and tubulin in H/R group were significant lower than those in normal group, but there was no significant difference between H/R group and EPO group. Conclusions The damage of cytoskeleton during ischemia/reperfusion may be time-dependent. EPO has no beneficial effect on the cytoskeleton after I/R injury.