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目的:观察大鼠脑缺血再灌注后海马内Nemo样激酶(Nemo-like kinase,NLK)蛋白表达的变化及细胞定位。方法:60只成年雄性Sprague-Dawley大鼠随机分为假手术组和脑缺血再灌注组,其中脑缺血再灌注组依术后不同的检测时间点分为:1、4、8 h和1、3、7 d 6个亚组。采用中脑动脉闭塞法(middle cerebral artery occlusion,MCAO)建立脑缺血再灌注模型,用Westernblot方法检测脑缺血再灌注后大鼠海马组织中NLK,活化的Caspase-3蛋白的变化趋势,用免疫组化和免疫荧光来检测NLK蛋白在脑组织中的细胞定位及可能发挥的生物学行为。结果:在脑缺血再灌注的海马组织中NLK蛋白随着脑缺血再灌注时间的延长而呈现先增高后降低,后再升高的趋势,其中缺血再灌注后8 h NLK蛋白表达达到高峰,与假手术组相比,差异有统计学意义(P﹤0.05)。活化的Caspase-3的表达随着观察时间的延长递增,3 d达到峰值,与假手术组比较,差异有统计学意义(P﹤0.05),随后下降。NLK定位于脑组织中的神经元细胞尤其是海马CA1区的锥体神经元。结论:脑缺血再灌注后NLK蛋白的表达显著上调,提示NLK参与了成年大鼠脑缺血的发展。
Objective: To observe the change of nemo-like kinase (NLK) protein expression and cell localization in the hippocampus after cerebral ischemia-reperfusion in rats. Methods: Sixty adult male Sprague-Dawley rats were randomly divided into sham-operated group and cerebral ischemia-reperfusion group. The cerebral ischemia-reperfusion group was divided into four groups 1,3,7 d 6 subgroups. Cerebral ischemia-reperfusion model was established by middle cerebral artery occlusion (MCAO). The changes of NLK and activation of Caspase-3 protein in hippocampus of rats after cerebral ischemia-reperfusion were detected by Western blot. Immunohistochemistry and immunofluorescence were used to detect the cellular localization of NLK protein in brain tissue and possible biological behavior. Results: In the hippocampus of rats with cerebral ischemia and reperfusion, the expression of NLK protein increased first and then decreased and then increased with the time of cerebral ischemia-reperfusion. The expression of NLK protein reached the peak at 8 h after ischemia-reperfusion Peak, compared with the sham operation group, the difference was statistically significant (P <0.05). The expression of activated Caspase-3 increased with the prolongation of observation time and reached the peak at 3 days. The difference was statistically significant (P <0.05) compared with the sham operation group, and then decreased. NLK is localized in neuronal cells in brain tissue, especially pyramidal neurons in the hippocampal CA1 region. Conclusion: The expression of NLK protein is significantly up-regulated after cerebral ischemia-reperfusion, suggesting that NLK is involved in the development of cerebral ischemia in adult rats.