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中国应用生理学杂志 ›› 2018, Vol. 34 ›› Issue (3): 277-282.doi: 10.12047/j.cjap.5627.2018.065

• 研究论文 • 上一篇    下一篇

脑缺血和缺血再灌注大鼠皮层神经元自噬的动态变化

陶红苗1, 单小云2, 李旭升1, 陈浩浩1, 毛宇飞1, 何忠平3   

  1. 1. 金华职业技术学院, 浙江 金华 321000;
    2. 金华市中心医院, 浙江 金华 321000;
    3. 金华市食品药品检验检测研究院, 浙江 金华 321000
  • 收稿日期:2017-08-28 修回日期:2018-02-02 出版日期:2018-05-28 发布日期:2018-09-08
  • 通讯作者: 陶红苗,Tel:0579-82232590,13516960975;E-mail:thm761023@163.com E-mail:thm761023@163.com
  • 基金资助:
    浙江省科技厅公益技术应用研究计划项目(2015C37118);金华市科技局社会发展类项目(2015-3-057)

Dynamic changes of autophagy activity of cortical neurons in rats with cerebral ischemia and cerebral ischemia/reperfusion

TAO Hong-miao1, SHAN Xiao-yun2, LI Xu-sheng1, CHEN Hao-hao1, MAO Yu-fei1, HE Zhong-ping3   

  1. 1. Jinhua Polytechnic, Jinhua 321000, China;
    2. Jinhua Central Hospital, Jinhua 321000, China;
    3. Jinhua Inspection and Research Institute for Food and Drug Control, Jinhua 321000, China
  • Received:2017-08-28 Revised:2018-02-02 Online:2018-05-28 Published:2018-09-08
  • Supported by:
    浙江省科技厅公益技术应用研究计划项目(2015C37118);金华市科技局社会发展类项目(2015-3-057)

摘要: 目的:探讨脑缺血和缺血/再灌注不同时间大鼠大脑皮层神经元自噬的变化。方法:健康雄性SD大鼠60只,随机分为:假手术(Sham)组(n=10),脑缺血和缺血/再灌注模型组(n=50).模型组分别在缺血30min、2h,缺血2h再灌注1h、6h、24h五个时间点,随机抽取10只大鼠,测定脑梗死体积和脑含水量,同时采用Western印迹法测定各组大鼠大脑皮层中微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)的水平,透射电镜检测大脑皮层神经细胞自噬情况。结果:脑缺血30min时LC3-Ⅱ/Ⅰ比值未见明显上升,缺血2h时LC3-Ⅱ/Ⅰ比值开始升高,明显高于Sham组(P<0.01);缺血/再灌注1h、6h时LC3-Ⅱ/Ⅰ比值虽较缺血2h组有所下降,但仍明显高于Sham组(P<0.05);缺血/再灌注24h时LC3Ⅱ/Ⅰ比值达高峰,明显高于Sham组(P<0.01)。透射电镜观察进一步证实该现象。缺血/再灌注6h和24h时大鼠脑梗死体积明显增加,与Sham组比较有统计学差异(P<0.01)。缺血/再灌注24h大鼠脑组织含水量明显增加,明显高于Sham组(P<0.05)。HE染色显示:仅在缺血/再灌注24h组大鼠皮层见组织水肿、疏松,部分细胞变性、凋亡,海马区见大量神经元细胞核皱缩、深染呈变性凋亡状。结论:局灶性脑缺血和缺血/再灌注模型中大脑皮层缺血2 h神经元自噬即明显激活,缺血/再灌注1 h、6 h自噬均持续增高,缺血/再灌注24 h自噬达高峰。

关键词: 脑, 缺血再灌注损伤, LC3, 自噬, 皮层神经元, 大鼠

Abstract: Objective: To investigate the changes of autophagy in cerebral cortex neurons at different times after cerebral ischemia and ischemia/reperfusion (I/R) in rats.Methods: Healthy male SD rats (n=60) were randomly assigned into sham-operation group (sham, n=10), cerebral ischemia and ischemia/reperfusion model group (I/R, n=50). In the model group, 10 rats were randomly sampled at five different time points:ischemia 30 min, 2 h, and reperfusion 1 h, 6 h and 24 h after ischemia 2 h. Cerebral infarction volume and cerebral water content were measured. Autophagy of cerebral cortical neurons was examined by transmission electron microscopy (TEM). The protein levels of microtubule associated protein light chain 3-Ⅱ (LC3-Ⅱ) in cerebral cortex of the rats in each group were determined by Western blot.Results: LC3-Ⅱ/Ⅰ ratio had no obvious rise at ischemia 30min,and it began to increase at ischemia 2 h point which was significantly higher than that in sham group (P<0.01).LC3-Ⅱ/Ⅰ ratio was still higher at ischemia/reperfusion 1h and 6h points than that in sham group (P<0.05),although it was decreased comparison with that at ischemia 2 h point. LC3-Ⅱ/Ⅰ ratio reached a peak at ischemia/reperfusion 24 h point, which was significantly higher than that in sham group (P<0.01). This phenomenon was further confirmed by the transmission electron microscopy. The infarction volume at ischemia/reperfusion 6h and 24 h points was increased significantly, and there was a statistical difference compared with sham group (P<0.01). The content of water in the brain tissue of rats at ischemia/reperfusion 24 h was increased obviously, which was significantly higher than that in sham group (P<0.05). Edema, rarefaction, some cell degeneration and apoptosis only in the cortical tissue of rats at ischemia/reperfusion 24 h were showed,and a large number of neurons nuclei in the hippocampus was shrunken,hyperchromatic showing degeneration and apoptosis by HE staining.Conclusion: Neurons autophagy in cerebral cortices of rats with focal cerebral ischemia and ischemia/reperfusion was obviously activated at ischemia 2 h point, it was increased continuously at ischemia/reperfusion 1 h and 6 h points and reached the peak at ischemia/reperfusion 24 h point.

Key words: brain, ischemic/reperfusion injury, LC3, autophagy, cortical neurons, rat

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