摘要
目的观察大鼠永久性局灶脑缺血皮质缺血半暗带区(IP)磷酸化应激活化激酶/C-Jun氨基末端激酶(P-SAPK/JNK)及原癌基因c-Myc mRNA转录的表达情况,探讨IP区神经细胞凋亡的可能机制。方法健康雄性Sprague-Dawley大鼠72只,随机分为假手术组和永久性大脑中动脉阻塞(pMCAO)后1、3、6、12、24 h组,每组12只。线栓法制备大鼠pMCAO模型,提取缺血不同时间点大鼠脑皮质IP区的组织。应用原位细胞凋亡(TUNEL)检测法分析IP区皮质神经元的凋亡情况,采用免疫组织化学法分析脑缺血后P-SAPK/JNK细胞的表达情况,采用Western-blot检测P-SAPK/JNK蛋白含量;采用实时定量PCR技术检测c-Myc mRNA的转录水平。结果①大鼠pMCAO后3 h,IP区TUNEL阳性细胞和P-SAPK/JNK阳性细胞均明显增加,12 h达高峰,与假手术组比较,差异有统计学意义,P<0.01。②Western-blot结果证明,大鼠pMCAO后3 h,P-SAPK/JNK蛋白水平增加,12 h达高峰,各时间点与假手术组比较,差异有统计学意义,P<0.01。③实时定量PCR结果表明,大鼠pMCAO后6 h,IP区c-Myc mRNA的转录水平明显上调,12 h达高峰,均高于假手术组,差异有统计学意义,P<0.01。④IP区皮质P-SAPK/JNK蛋白水平与神经元凋亡及c-Myc mRNA转录水平均呈正相关系(r=0.985,P<0.01;r=0.887,P<0.05)。结论 pMCAO模型IP区神经元的凋亡可能与JNK/c-Myc信号通路的激活有关。
Objectives To observe the transcriptional expression of the phospho-stress activated protein kinase/Jun N-terminal kinase（P-SAPK/JNK） and proto-oncogene proteins c-Myc mRNA in cortical ischemic penumbra（IP） after permanent focal cerebral ischemia in rats and to investigate the possible mechanism of neuronal apoptosis in IP.Methods Seventy-two healthy male Sprague-Dawley rats were randomly allocated into sham operation group,1-,3-,6-,12-,and 24-hour after permanent middle cerebral artery occlusion（pMCAO） groups（n=12 in each group）.The pMCAO model was induced in rats.The rat cerebral cortical tissue in IP at different time points after ischemia was extracted.In situ apoptosis（TUNEL） assay was used to analyze the cortical neuronal apoptosis in IP.Immunohistochemical method was used to analyze the expression of P-SAPK/JNK after cerebral ischemia.Western blot analysis was used to detect the level of P-SAPK/JNK.Real-time PCR technology was used to detect the transcriptional level of c-Myc mRNA.Results ①The numbers of TUNEL positive cells and P-SAPK/JNK-positive cells in IP area were increased significantly at 3 hours after pMCAO in rats.They reached the peak at 12 hours.Compared to the sham operation group,there were significant differences（P0.01）.②The results of Western blot showed that the level of P-SAPK/JNK increased after 3-hour pMCAO in rats.It reached the peak at 12 hours.There were significant differences compared to the sham operation group at each time point（P0.01）.③The results of the real-time PCR showed that the transcriptional level of c-Myc mRNA at 6 hours after pMCAO in rats was up-regulated obviously.It reached the peak at 12 hours and was higher than that in the sham operation group.There was significant difference（P0.01）.The level of cortical P-SAPK/JNK in IP was positively correlated with the neuronal apoptosis and the transcriptional level of c-Myc mRNA（r=0.985,P0.01;r=0.887,P0.05）.Conclusion The neuronal apoptosis in the ischemic penumbra after permanent MCA occlusion may be associated with the activation of JNK/c-Myc signaling pathway.
出处
《中国脑血管病杂志》
CAS
2012年第3期136-141,共6页
Chinese Journal of Cerebrovascular Diseases
基金
上海市普陀区卫生科技研究支持项目(PWG-06-01)
