p38MAPK蛋白在体外培养神经元中的表达及W-7的干预作用

J4 ›› 2011, Vol. 37 ›› Issue (1): 47-50.

p38MAPK蛋白在体外培养神经元中的表达及W-7的干预作用

刘学文¹, 胥向红², 郎森阳³, 蔡爱民¹

（1.辽宁医学院附属第一医院神经内科|辽宁锦州121001；2.辽宁医学院附属第三医院神经内科|辽宁锦州121000；3.解放军总医院神经内科|北京100853）

收稿日期:2010-04-30 出版日期:2011-01-28 发布日期:2011-01-28
通讯作者: 刘学文 E-mail:sjnklxw@126.com
作者简介:刘学文(1969-)|女|辽宁省义县人|副主任医师|医学博士|主要从事癫痫的基础及临床研究。

Expression of p38MAPK in cultured cortical neurons in vitro and intervention effect of W-7

LIU Hua-Wen¹, XU Xiang-Hong², LANG Sen-Yang,³ CAi Ai-Min¹

(1.Department of Neurology,First Affiliated Hospital, Liaoning Medical College. Jinzhou 121001,China|2.Department of Neurology,Third Affiliated Hospital, Liaoning Medical College,Jinzhou 121000,China;3.Depatment of Neurology,General Hospital of PLA,Beijing 100853,China)

Received:2010-04-30 Online:2011-01-28 Published:2011-01-28
Supported by:
辽宁省科技厅博士启动基金资助课题(20091049)；辽宁医学院附属第一医院博士启动基金资助课题(20090812)

摘要/Abstract

摘要：

目的:探讨p38MAPK蛋白在体外培养神经元中的激活及钙调蛋白抑制剂W-7的干预作用。方法：神经元原代培养并采用NSE免疫细胞化学技术鉴定神经元。取原代培养7 d的大鼠皮质神经元随机分为5组：正常对照组，仅用DMEM/F12完全培养基；NMDA组，去除正常神经元培养液，加入NMDA 50 μmol.L^-1，处理时间10 min；W-7干预组，W-7浓度分别为25、50和100 μmol.L^-1，继续培养24 h，加入NMDA　50 μmol.L^-1。免疫细胞化学染色法及免疫印记法检测皮质神经元内p38MAPK蛋白的表达水平。结果：培养6～12 h后大部分神经元贴壁，随时间延长形态多变，突起逐渐增多，神经元突起间互相接触形成网络。NSE鉴定神经元纯度为90.86%；免疫细胞化学染色法检测，NMDA组p38MAPK蛋白表达水平高于对照组(P<0.05)，W-7组低于NMDA组(P<0.05或P<0.01)，呈剂量依赖性；Western blotting法检测，NMDA组p38MAPK蛋白表达水平高于对照组（P<0.05），W-7干预组p38 MAPK表达水平低于NMDA组（P<0.01），呈剂量依赖性。结论：NMDA导致皮质神经元p38MAPK蛋白表达增高，W-7可降低p38MAPK的蛋白表达。

关键词: p38丝裂原活化蛋白激酶类；神经元；N-甲基天冬氨酸；大鼠, Sprague-Dawley;细胞培养

Abstract:

Abstract:Objective
To investigate the activation of p38MAPK protein in cultured cortical neurons after NMDA injury and intervention effect of W-7. Methods The neurons were identified by polyclonal antibody against neuron specific enolase (NSE).The primary cortical neurons cultured for 7 d were randomly divided into 5 groups:control group;NMDA-injured group;three-doses of W-7 pretreatment groups. The cortical neurons were pre-cultured with regular media including different doses of W-7 respectively as 25,50 and 100 μmol.L^-1 for 24 h before exposed to NMDA (50 μmol.L^-1). The p38MAPK protein expression in cultured cortical neurons was detected by immunocytochemiscal staining and Western blotting. Results A large number of hippocampal neurons began to adhere to cover the glasses 6-12 h after culture. They showed different shapes after clinging to the plate. Their processes connected into nets and they were different in length and thickness. The proportion of positive neurons was 90.86%.Compared with control group,the expression of p38MAPK in cultured neuron was remarkably up-regulated in NMDA injured group by immunocytochemistrical staining and up-regulated in NMDA injured group by Western blotting (P<0.05),it was significantly down-regulated in W-7 pretreatment groups compared with NMDA group (P<0.05 or P<0.01). Conclusion NMDA could up-regulate the expressin of p38MAPK in cultured cotical neurons,and W-7 could down-regulated the expression of p38MAPK.

Key words: p38 mitogen-activated protein kinases;neurons;N-methylaspartate, rats,Spragne-Dawley;cell culture

中图分类号:

R338.1

刘学文, 胥向红, 郎森阳, 蔡爱民. p38MAPK蛋白在体外培养神经元中的表达及W-7的干预作用[J]. J4, 2011, 37(1): 47-50.

LIU Hua-Wen, XU Xiang-Hong, LANG Sen-Yang, CAi Ai-Min. Expression of p38MAPK in cultured cortical neurons in vitro and intervention effect of W-7[J]. J4, 2011, 37(1): 47-50.