摘要
目的探讨缺血再灌流脑区半胱氨酸蛋白酶-3(caspase-3)活性变化与神经元凋亡的关系.方法采用Western Blotting、原位细胞凋亡检测和免疫组化染色等方法,观察大鼠大脑中动脉栓塞2 h后再灌流1、6、12、24 h时,颞顶叶皮层缺血脑区caspase-3表达和活化、多(ADP-核糖)聚合酶(PARP)表达和切割灭活及神经元凋亡程度的变化.结果再灌流1、6、12及24 h组,缺血脑区的caspase-3前体及其12 000切割片段含量的相对灰度值分别为16.72±2.96、28.36±3.51、51.15±3.10、76.14±3.45及8.17±2.31、15.36±1.39、31.23±5.43、58.95±6.28;PARP及其24 000切割片段含量的相对灰度值分别为12.63±3.02、22.65±4.38、30.81±3.16、67.49±8.59及6.02±0.73、12.86±2.30、20.76±3.16、27.36±2.63;PARP阳性神经元及凋亡神经元的密度(细胞数/0.1 mm2)分别为68.00±6.67、91.40±9.56、112.20±11.78、127.00±11.51及83.31±7.53、100.70±6.16、121.53±7.21、197.36±11.78.以上6种指标的变化彼此间均呈正相关(r= 0.805～0.942, P<0.01);除6 h与12 h组间PARP含量的差异及6 h与12 h组间和12 h与24 h组间PARP阳性神经元密度的差异无统计学意义外,上述6种指标的其余组间差异均有统计学意义(P<0.05～0.01).结论再灌流可迅速增加缺血脑区caspase-3表达和活化,后者通过切割灭活PARP启动受损神经元凋亡;再灌流早期PARP表达适量增加可部分抵消上述过程,保护受损神经元;PARP过度表达和蓄积可能会通过干扰能量代谢和启动其他凋亡途径加速受损神经元死亡.
Objective To investigate the relationship between the variations of expression and activity of caspase-3, and the neuronal apoptosis in cerebral injured region after forebrain ischemia-reperfusion.Methods The rats of middle cerebral artery occlusion and reperfusion were used as the research model. The expression and activation of caspase-3, expression and cleavage of poly (ADP-ribose) polymerase (PARP), and neuronal apoptosis in the temporal-parietal-cortex regions injured by ischemia for 2 hours, then reperfusions at 1, 6, 12, 24 hour points were studied using Western blotting, in situ apoptotic detection (TUNEL method) and immunohistochemistry. The correlations among their variations were also analyzed.Results In cerebral ischemic regions reperfused for 1, 6, 12, 24 hour, relative gray values of the contents of procaspase-3 and its 12 kd cleavage fragment were 16.72±2.96, 28.36±3.51, 51.15±3.10, 76.14±3.45 and 8.17±2.31, 15.36±1.39, 31.23±5.43, 58.95±6.28 respectively; the relative gray values of PARP and its 24 kd cleavage fragment were 12.63±3.02, 22.65±4.38, 30.81±3.16, 67.49±8.59 and 6.02±0.73, 12.86±2.30, 20.76±3.16, 27.36±2.63 respectively; densities (cell number/0.1mm2) of neurons expressing PARP were 68.00±6.67, 91.40±9.56, 112.20±11.78,127.00±11.51 and the ones of apoptotic neurons were 83.31±7.53, 100.70±6.16, 121.53±7.21, 197.36±11.78 respectively. The variations of above six parameters were all correlated positively with one another (r=0.805~0.942, P<0.01) and all of them were increased relevantly along with prolongation of the reperfusion time. Every one of the differences, except that between PARP contents at 6 and 12 hours after reperfusion as well as those between PARP positive neuron densities at 6 and 12 hours, and 12 and 24 hours after reperfusion, had statistical significance (P<0.05~0.01).Conclusions The reperfusion might rapidly increase the expression and activation of caspase-3 in cerebral ischemic regions. The activated caspase-3 might promote the injured-neuron apoptosis by cleaving and inactivating PARP. During the early stage of the reperfusion, appropriate increase of the PARP expression could possibly protect the injured neurons by partly offsetting above role of the caspase-3. However, the excessive expression and accumulation of PARP might possibly accelerate the death of the injured neurons by interfering the energy metabolism and switching on the other apoptotic pathway.
出处
《中华神经科杂志》
CAS
CSCD
北大核心
2005年第1期25-29,共5页
Chinese Journal of Neurology
基金
天津市科委重点课题基金资助项目(953105711)
