Cdk5与大鼠脑缺血再灌注损伤时细胞凋亡的关系

Cyclin Dependent Kinase 5(Cdk5) Promotes Apoptosis in Ischemic Cerebral Tissue after Reperfusion Injury in Rats

目的利用大鼠可逆性大脑中动脉栓塞模型(MCAO),研究周期素依赖性蛋白激酶5(cyclin dependent kinase 5,Cdk5)对视网膜母细胞瘤蛋白(Rb)的磷酸化作用与细胞凋亡的关系。方法线栓法制作大鼠MCAO模型,随机分为再灌注0、3、6、9、24h组。通过免疫组化染色观察缺血侧大脑Cdk5和磷酸化Rb的数量和变化,TUNEL标记法检测凋亡神经元数量和变化,Westernblot检测不同再灌注时间Cdk5蛋白水平的表达。结果缺血侧Cdk5随再灌注时间增加而增加,24h达到高峰;再灌注6h出现磷酸化的视网膜母细胞瘤蛋白(pRb),并随再灌注时间增加而增加,24h达到高峰。缺血侧凋亡阳性细胞变化趋势与Cdk5基本一致。缺血侧Cdk5蛋白的表达随再灌注时间增加而增加,缺血对侧无明显变化。结论大鼠局限性脑缺血再灌注损伤可以诱导Cdk5数量和激酶活性增加,通过底物磷酸化作用引起神经细胞凋亡。

Objective To investigate the relationship between the phosphorylation effect of Cdk5 on retinoblastoma （Rb） protein and neuron apoptosis in rats with a reversible middle cerebral artery occlusion （MCAO）. Methods An intraluminal MCAO rat model was set up. The rats were randomly divided into five groups according to different reperfusion time points at 0,3, 6,9, 24 h, respectively. Cdk5 and phosphorylated Rb, a Cdk5 substrate, were measured quantitatively in the ischemic infarct hemisphere,using immunostaining with anti-Cdk5 antibody and anti-phospho-Rb antibody, respectively. With TUNEL staining, apoptotic cells were measured quantitatively with an imaging analysis system. Protein level of Cdk5 was detected by Western blot. Result In the ischemic hemisphere, the number of Cdk5 positive cells increased following the reperfusion time and reached a peak level at 24 h reperfusion, pRb positive cells appeared at 6 h reperfusion time, increased following the reperfusion time and got a peak at 24 h. Apoptotic cells increased following the reperfusion time and reached a peak ievel at 24 h reperfusion. The expression of Cdk5 increased following the reperfusion time. Conclusion Local cerebral ischemia reperfusion injury can induce an increased amount and enzyme activity of Cdk5 in rats. The phosphorylation of Cdk5 on its substrates might result in more neuron apoptosis in the penumbra of infarct brain.