吉林大学学报(医学版) ›› 2017, Vol. 43 ›› Issue (01): 47-51.doi: 10.13481/j.1671-587x.20170110

• 基础研究 • 上一篇    下一篇

匹鲁卡品致小鼠癫痫神经元模型的建立及其F-actin、Calponin 3和ROCK2的表达

古小云1, 张舒岩2, 袁羽帆1, 杨立彬3, 李艳超1, 李树蕾1   

  1. 1. 吉林大学基础医学院组织学与胚胎学系, 吉林 长春 130021;
    2. 吉林大学第一医院神经外科, 吉林 长春 130021;
    3. 吉林大学第一医院儿科, 吉林 长春 130021
  • 收稿日期:2016-09-02 出版日期:2017-01-28 发布日期:2017-02-08
  • 通讯作者: 李树蕾,副教授,硕士研究生导师(Tel:0431-85619477,E-mail:lsl@jlu.edu.cn) E-mail:lsl@jlu.edu.cn
  • 作者简介:古小云(1992-),女,重庆市人,在读医学硕士,主要从事神经系统发作性疾病发生机制方面的研究。
  • 基金资助:

    吉林省科技厅自然科学基金资助课题(20130101138JC);吉林大学白求恩医学科研支持计划青年科研基金资助课题(2069)

Establishment of mouse epileptic neuron model induced by pilocarpine and expressions of F-actin, Calponin 3 and ROCK2

GU Xiaoyun1, ZHANG Shuyan2, YUAN Yufan1, YANG Libin3, LI Yanchao1, LI Shulei1   

  1. 1. Department of Histology and Embryology, School of Basic Medical Sciences, Jilin University, Changchun 130021, China;
    2. Department of Neurosurgery, First Hospital, Jilin University, Changchun 130021, China;
    3. Department of Pediatrics, First Hospital, Jilin University, Changchun 130021, China
  • Received:2016-09-02 Online:2017-01-28 Published:2017-02-08

摘要:

目的:探讨匹鲁卡品所致小鼠癫痫神经元中细胞骨架丝状肌动蛋白(F-actin)、调宁蛋白3(Calponin 3)和Rho相关含卷曲螺旋蛋白激酶2(ROCK2)的表达,研究癫痫发作后RhoA/ROCK2通路与Calponin 3和F-actin解聚和重构之间的关系。方法:采用木瓜蛋白酶消化法分离ICR新生小鼠乳鼠皮层神经元,生长至第7天时用微管关联蛋白2(MAP2)抗体进行免疫组织化学染色鉴定神经元。将培养7d的神经元分为对照组和癫痫模型组,对照组用神经元培养基培养,癫痫模型组在神经元培养基中分别加入终浓度为2、3和4 mmol·L-1匹鲁卡品,24h后换为正常培养基。分别在造模后不同时间点取出各组细胞固定,进行免疫组织化学染色和荧光染色。采用Alex-546标记的phalloidin进行荧光染色观察神经元中F-actin分布;免疫组织化学染色观察神经元中Calponin 3、ROCK2和磷酸化ROCK2(p-ROCK2)表达和分布。结果:光镜下观察和F-acitn荧光染色,与对照组比较,造模24h后,2 mmol·L-1匹鲁卡品组细胞无明显变化;3 mmol·L-1匹鲁卡品组神经元中F-acitn出现解构,神经元部分突起消失,但在更换正常培养基后,细胞形态和F-actin结构在造模7d后逐渐恢复;4 mmol·L-1匹鲁卡品组神经元中F-actin结构崩解破坏严重,神经元突起不可逆性消失。免疫组织化学染色,对照组Calponin 3和ROCK2弥散分布于胞质中;在造模1d时癫痫模型组(3 mmol·L-1匹鲁卡品)神经细胞中Calponin 3和ROCK2明显聚集在细胞膜下方,随培养时间延长其表达量明显上调,位于细胞膜下方的p-ROCK2表达量也较对照组明显增加,同样随培养时间延长其表达量逐渐升高。结论:利用3 mmol·L-1匹鲁卡品成功建立癫痫神经元模型。3 mmol·L-1匹鲁卡品可使神经元细胞内ROCK2活化为p-ROCK2,并上调神经元内的ROCK2和p-ROCK2表达量,促使F-actin解聚导致神经元突起部分消失,p-ROCK2激活Calponin3磷酸化,促进F-actin重构和神经元形态结构的恢复。

关键词: 癫痫, 神经元, Rho相关含卷曲螺旋蛋白激酶2, 匹鲁卡品, 细胞骨架丝状肌动蛋白, 调宁蛋白3

Abstract:

Objective: To discuss the changes of expressions of filamentous actin(F-actin),Calponin 3 and Rho-associated coiled-coil-containing protein kinase 2(ROCK2) in neurons of the epileptic mice induced by pilocarpine, and to study the relationship among RhoA/ROCK2 pathway activation and Calponin 3 and the depolymerization and rearrangement of F-actin.Methods: The cortical neurons from ICR neonatal mice were isolated by using papain digestion and were identified with immunohistochemistry staining using microtubule associated protein 2(MAP2) antibody on the 7th day.The neurons cultured for 7 d were divided into control and epilepsy groups. The neurons in control group were cultured in normol neurobasal-A medium, and the neurons in epilepsy groups were cultured in medium with final concentrations of 2,3,and 4 mmol·L-1 of pilocarpine respectively which were replaced by normol neurobasal-A medium after 24 h. The neurons of mice in various groups were fixed at different time points after modeling, then immunohistochemistry and fluorescence staining were performed. The fluorescence intensity of F-actin in neurons was observed by phalloidine staining labeled by Alex-546, as well as the expressions and distributions of Calponin3, ROCK2 and p-ROCK2 in neurons were observed by immunohistochemical staining.Results: The results of light microscope observation and F-acitn fluorescence staining showed that the neurons in 2 mmol·L-1 pilocarpine group had no obviously changes 24 h after modeling compared with control group;the F-actin deconstruction and neurite disappearance partly of neurons in 3 mmol·L-1 pilocarpine group were found, which gradually restored in next 7 d after the culture medium was replaced by normal one;the severel and irreversibe damage of F-actin structure and neurite disappearance in 4 mmol·L-1 pilocarpine group were found.The results of immunohistochemical staining showed that Calponin 3 and ROCK2 dispersively distributed in cytoplasm in control group;as for epilepsy model group(3 mmol·L-1 pilocarpine),Calponin 3 and ROCK2 obviously aggregated beneath the cell membrane 1 d after modeling, and their expression levels were gradually increased with the prolongation of culture time. The expressions of p-ROCK2 in epileptic groups were significantly higher than that in control group, and they were increased with the prolongation of culture time.Conclusion: The neuron epilepsy models are successfully established by using 3 mmol·L-1 pilocarpine. 3 mmol·L-1 pilocarpine may activate ROCK2 to p-ROCK2,and up-regulate the expression levels of ROCK2 and p-ROCK2 in neurons, so it can promote the depolymerization of F-actin and result in neurite disappearance partly. p-ROCK2 may activate Calponin 3 phosphorylation and promote the F-actin reconstruction and the restoration of the morphology of epileptic neurons gradually.

Key words: epilepsy, calponin 3, Rho-associated coiled-coil-containing protein kinase 2, neurons, pilocarpine, filamentous actin

中图分类号: 

  • R742.1