CAJ | 학술논문

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.
목적 탐토대서심폐복소후신경세포선립체통투성전환공(MPTP)변화재세포색소C석방중적작용급가능궤제.방법 이56지성년웅성Sprague Dawley(SD)대서건립질식가빙록화갑치심박취정/심폐복소(CA/CPR)동물모형,수궤(수궤수자법)분위7조,즉가수술조、자주순배회복(ROSC)후3 h,6 h,12 h,24 h,48 h,72 h조,매조8지,분별재각시상점단두처사대서,채용차속리심법분리제비대뇌피질내선립체균장.채용분광광도법측정선립체불동시상MPTP적개방정도,Western blot면역잡교법검측선립체세포색소C석방(CytC).채용SPSS 11.5 for Windows통계연건분석결과.결과 ROSC후6 h내신경세포MPTP개방정도보지저수평,6 h이후개시신속대량개방,12 h개방정도체도최대,24 h개방정도략유축소,지48 h개방정도재차가대,72 h우명현축소,여대조조상비교차이균구유통계학의의(P＜0.05혹P＜0.01).정상뇌조직선립체CytC단백재상대분자질량14 000처출현일강양성조대,이포장단백중CytC단백함량심미,정일약양성조대.ROSC후3 h선립체CytC단백미견현저변화,이포장성분중개시증다,지ROSC후24 h대부분CytC이석방지포장중,차시선립체조대명현감약.결론 대서CA/CPR병ROSC후신경MPTP적개방정도여CytC석방적량정명현적상관성,MPFP적개방촉사CytC적석방.
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.