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

发布时间：2018-05-07 17:31

本文选题：癫痫 + 神经元　；参考：《吉林大学学报(医学版)》2017年01期

【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4mmol·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,2mmol·L~(-1)匹鲁卡品组细胞无明显变化;3mmol·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;4mmol·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(3mmol·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3mmol·L~(-1)匹鲁卡品成功建立癫痫神经元模型。3mmol·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Calponin 3磷酸化,促进F-actin重构和神经元形态结构的恢复。
[Abstract]:Objective: to investigate the expression of cytoskeletal filamentous actin F-actinine (3(Calponin _ 3) and Rho associated spiroprotein kinase _ 2 (ROCK2) in mouse epileptic neurons induced by pilocarpine. To study the relationship between RhoA/ROCK2 pathway and Calponin 3 and F-actin depolymerization and remodeling after seizure. Methods: the cortical neurons of newborn ICR mice were isolated by papain digestion. The neurons were identified by immunohistochemical staining with microtubule-associated protein 2MAP2) antibody at the 7th day of growth. The neurons cultured for 7 days were divided into control group and epileptic model group. The control group was cultured on neuron medium. In the epileptic model group, the final concentration of 2t3 and 4mmol Lnka-1 were added to the neuron culture medium for 24 hours and then replaced into the normal medium. The cells were fixed at different time points and stained by immunohistochemistry and fluorescence staining. Alex-546 labeled phalloidin was used to observe the distribution of F-actin in neurons, and immunohistochemical staining was used to observe the expression and distribution of Calponin _ 3 rock _ 2 and phosphorylated rock _ 2o _ (p-ROCK2) in neurons. Results: under light microscope and F-acitn fluorescence staining, compared with the control group, there was no obvious change of F-acitn in the neurons of the 2 mmol / L ~ (-1) pilocarpine group after 24 hours of model making, and the neurite disappeared in the neurons of the pilocarpine group, but after replacing the normal culture medium, there was no significant change in the number of F-acitn in the neurons, but after the replacement of the normal culture medium, there was no significant change in the cells in the model group. Cell morphology and F-actin structure gradually recovered after 7 days of modeling. In Pilucapine group, the F-actin structure collapsed seriously, and the neurite irreversibly disappeared. Immunohistochemical staining showed that Calponin _ 3 and ROCK2 were diffused in cytoplasm in control group, and Calponin _ 3 and ROCK2 in epileptic model group (n = 3 mmol L ~ (-1) ~ (-1) pilocarpine on day 1, respectively. The expression of Calponin _ 3 and ROCK2 were significantly increased with the prolongation of culture time, and the expression of Calponin _ 3 and ROCK2 were significantly increased with the prolongation of culture time. The expression of p-ROCK2 under the cell membrane was also significantly higher than that in the control group, and the expression level increased gradually with the extension of culture time. Conclusion: the model of epileptic neuron. 3 mmol / L ~ (-1) Pilucapine can activate ROCK2 into p-ROCK2, and up-regulate the expression of ROCK2 and p-ROCK2 in neurons. F-actin depolymerization induced partial disappearance of neuronal processes and p-ROCK2 activation of Calponin _ 3 phosphorylation, F-actin remodeling and neuronal morphological recovery.
【作者单位】：吉林大学基础医学院组织学与胚胎学系;吉林大学第一医院神经外科;吉林大学第一医院儿科;
【基金】：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)
【分类号】：R742.1;R-332

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