大鼠心肺复苏后神经细胞线粒体通透性转换孔变化在细胞色素C释放中的作用  

Role of the permeability transition pore in cytochrome C release from mitochondria in rats brain after resuscitation from cardiac arrest

作　　者：马宇洁[1] 杨兴易[2] 林兆奋[2] 缪明永[3] 张雷[4] 宁波[1] 

机构地区：[1]空军总医院重症监护科,北京100142 [2]第二军医大学第二附属医院急救科 [3]第二军医大学基础部生物化学与分子生物学教研室 [4]乌鲁木齐总医院急诊科

出　　处：《中华急诊医学杂志》2010年第10期1015-1018,共4页Chinese Journal of Emergency Medicine

基　　金：国家自然科学基金资助项目（30500173）

摘　　要：目的 探讨大鼠心肺复苏后神经细胞线粒体通透性转换孔（MPTP）变化在细胞色素C释放中的作用及可能机制.方法 以56只成年雄性Sprague Dawley（SD）大鼠建立窒息加冰氯化钾致心搏骤停/心肺复苏（CA/CPR）动物模型,随机（随机数字法）分为7组,即假手术组、自主循环恢复（ROSC）后3 h,6 h,12 h,24 h,48 h,72 h组,每组8只,分别在各时相点断头处死大鼠,采用差速离心法分离制备大脑皮质内线粒体匀浆.采用分光光度法测定线粒体不同时相MPTP的开放程度,Western blot免疫杂交法检测线粒体细胞色素C释放（CytC）.采用SPSS 11.5 for Windows统计软件分析结果.结果 ROSC后6 h内神经细胞MPTP开放程度保持低水平,6 h以后开始迅速大量开放,12 h开放程度达到最大,24 h开放程度略有缩小,至48 h开放程度再次加大,72 h又明显缩小,与对照组相比较差异均具有统计学意义（P＜0.05或P＜0.01）.正常脑组织线粒体CytC蛋白在相对分子质量14 000处出现一强阳性条带,而胞浆蛋白中CytC蛋白含量甚微,呈一弱阳性条带.ROSC后3 h线粒体CytC蛋白未见显著变化,而胞浆成分中开始增多,至ROSC后24 h大部分CytC已释放至胞浆中,此时线粒体条带明显减弱.结论 大鼠CA/CPR并ROSC后神经MPTP的开放程度与CytC释放的量呈明显的相关性,MPFP的开放促使CytC的释放.Objective To analyse the relationship between cytochrome C release and the opening degree of the permeability transition pore （PTP） during in post cardiopulmonary resuscitation（CPR） rats. Method Adult male Sprague Dawley （SD）rats were randomly （random number ） divided into a surgical sham group （no CA/CPR） （ n = 8） and CA/CPR group （ n = 48）. Animals in CA/CPR group was induced by asphyxiation and icecold 0.5 mmol/L KCI and killed by decapitation and processed for isolation of brain cortex mitochondria at 3 h,6 h,12 h,24 h,48 h,72 h after restoration of spontaneous circulation （ROSC）. MPTP opening degree was based on the absorbance changes of the mitochondrial suspension at 540 nm. Western blot analysed the release of CytC from mitochondrial to cytoplast. Results Neural cells MPTP always remained opening post ROSC. The opening degree of MPTP was not reaching the peak instantly. While its＇ change depended on time： remaining low level within 6 h post ROSC,then rapidly opening, till 12 h reaching the largest degree,and at 24 h post ROSC slightly shrunken. All these suggested that mitochondria started to shink. While at 48 h the opening degree largen again, shrunken once more at 72 h,but not reaching the normal level （ P 〈 0.05 or P 〈 0.01）. Cytochrome C could not be detected in the cytosol in the earlier phases of the sham-operated brain samples but appeared in the mitochondria.The amount of cytochrome C released was proportional after restoration of spontaneous circulation 3 h and 24 h,failed to detect the enzyme in the mitochondria fraction. Conclusions We draw the conclusion MPTP plays a key role in neurocyte apoptosis after CA/CPR, which leads to mitochondria release CytC.

关 键 词：心肺复苏 心搏骤停 缺血.再灌注损伤 线粒体 线粒体通透性转换孔 细胞色素C 凋亡 大鼠 

分 类 号：R363[医药卫生—病理学]