经典型蛋白激酶Cγ及其相互作用蛋白14-3-3γ在原代培养小鼠脑皮层神经元氧-糖剥夺缺血损伤中的作用

doi:10.16098/j.issn.0529-1356.2015.02.001

解剖学报 ›› 2015, Vol. 46 ›› Issue (2): 145-150.doi: 10.16098/j.issn.0529-1356.2015.02.001

• 神经生物学 • 下一篇

经典型蛋白激酶Cγ及其相互作用蛋白14-3-3γ在原代培养小鼠脑皮层神经元氧-糖剥夺缺血损伤中的作用

章欣欣杨璇马维陈璐勔韩松李俊发*

首都医科大学神经生物学系，北京脑重大疾病研究院，北京 100069

收稿日期:2014-10-14 修回日期:2014-11-19 出版日期:2015-04-06 发布日期:2015-04-06
通讯作者: 李俊发 E-mail:ZHXX0903@163.COM

Role of conventional protein kinase Cγ and its interacting protein 14-3-3γ in oxygen\|glucose deprivation-induced ischemic injury of primary cultured mouse cortical neurons

ZHANG Xin-xin YANG Xuan MA Wei CHEN Lu-mian HAN Song LI Jun-fa*

Department of Neurobiology and Beijing Institute for Brain Disorders, Capital Medical University, Beijing 100069， China

Received:2014-10-14 Revised:2014-11-19 Online:2015-04-06 Published:2015-04-06
Contact: LI Jun-fa E-mail:ZHXX0903@163.COM

摘要/Abstract

摘要：

目的探讨经典型蛋白激酶Cγ (cPKCγ)及其相互作用蛋白14-3-3γ在原代培养小鼠脑皮层神经元氧-糖剥夺(OGD)缺血损伤中的作用。方法利用cPKCγ基因敲除(cPKCγ^-/-)小鼠和原代培养脑皮层神经元1h OGD/24h复糖复氧(R)模拟离体细胞缺血模型，给予14-3-3γ抑制剂R18干扰，采用免疫共沉淀(Co-IP)、蛋白免疫印迹(Western blotting)和四甲基偶氮唑盐(MTT)比色法，验证cPKCγ与14-3-3γ的相互作用及其对原代培养小鼠脑皮层神经元1h OGD/24h R缺血损伤的影响。结果 Co-IP结果证明，14-3-3γ与cPKCγ在小鼠脑皮层中确实存在相互作用；1h OGD/24h R处理使原代培养野生型(cPKCγ ^+/+)小鼠脑皮层神经元内cPKCγ蛋白水平明显降低，而14-3-3γ蛋白水平显著增加(P<0.05,每组n=6)；对于原代培养cPKCγ^-/-小鼠脑皮层神经元，尽管14-3-3γ表达水平明显高于cPKCγ^+/+小鼠脑皮层神经元，但1h OGD/24h R处理却使14-3-3γ表达水平显著降低(P<0.05, 每组n=6)，提示cPKCγ可影响神经元内14-3-3γ的表达水平。此外，MTT结果表明，14-3-3γ抑制剂R18 (100μmol/L)可显著加重1h OGD/24h R处理原代培养cPKCγ^-/-和cPKCγ^+/+小鼠脑皮层神经元的缺血损伤(P<0.05，每组n=6)。结论 cPKCγ与14-3-3γ在小鼠脑皮层神经元内存在相互作用并影响14-3-3γ的蛋白表达水平，cPKCγ和14-3-3γ在原代培养小鼠脑皮层神经元缺血/低氧损伤中起保护作用。

关键词: 原代培养小鼠脑皮层神经元, 氧-糖剥夺, 缺血损伤, 经典型蛋白激酶Cγ, 14-3-3γ, 免疫印迹法, 小鼠

Abstract:

Objective To explore the role of conventional protein kinase Cγ (cPKCγ) and its possible interacting protein 14-3-3γ in 1hour oxygen-glucose deprivation (OGD)/24hours reoxygenation and glucose restoration (R)-induced ischemic injury in primary cultured cortical neurons of mice.Methods Using cPKCγ gene knockout (cPKCγ^-/-) mice, 1hour OGD/24hours R-induced ischemic injury model of primary cultured cortical neurons, and the techniques of co-immunoprecipitation (Co-IP), Western blotting and 3-(4,5-dimethyl thiazole-2)-2,5-diphenyl four azole nitrogen bromine salt (MTT) assay, the interaction of cPKCγ and 14-3-3γ and their effects on ischemic injury were determined. Results The Co-IP results confirmed the existence of cPKCγ interaction with 14-3-3γ in the cerebral cortex of mice; the decreased protein expression of cPKCγ was accompanied with the increase of 14-3-3γ protein levels in primary cultured cortical neurons from cPKCγ+/+mice after 1hour OGD/24hours R treatment (P<0.05, n=6 per group). Although the 14-3-3γ protein level in cortical neurons from cPKCγ-/-mice was higher than that from cPKCγ+/+ mice, 1hour OGD/24hours R treatment caused a significant decrease of 14-3-3γ protein level in cortical neurons from cPKCγ-/-mice (P<0.05, n=6 per group), suggesting cPKCγ may affect 14-3-3γ protein expression level in cortical neurons. In addition, the MTT results demonstrated that the 14-3-3γ inhibitor R18 (100μmol/L) significantly enhanced 1hour OGD/24hours R-induced ischemic injury of primary cultured cortical neurons from both cPKCγ^+/+and cPKCγ^-/-mice (P<0.05, n=6 per group).Conclusion cPKCγ may interact with 14-3-3γ and affect 14-3-3γ protein expression level in cortical neurons of mice. cPKCγ and 14-3-3γ may protect primary cultured cortical neurons against 1hour OGD/24hours R-induced ischemic injury.

Key words: Primary cultured cortical neuron, Oxygen-glucose deprivation, Ischemic injury, Conventional protein kinase Cγ, 14-3-3γ, Western blotting, Mouse

章欣欣杨璇马维陈璐勔韩松李俊发*. 经典型蛋白激酶Cγ及其相互作用蛋白14-3-3γ在原代培养小鼠脑皮层神经元氧-糖剥夺缺血损伤中的作用[J]. 解剖学报, 2015, 46(2): 145-150.

ZHANG Xin-xin YANG Xuan MA Wei CHEN Lu-mian HAN Song LI Jun-fa*. Role of conventional protein kinase Cγ and its interacting protein 14-3-3γ in oxygen\|glucose deprivation-induced ischemic injury of primary cultured mouse cortical neurons[J]. AAS, 2015, 46(2): 145-150.

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经典型蛋白激酶Cγ及其相互作用蛋白14-3-3γ在原代培养小鼠脑皮层神经元氧-糖剥夺缺血损伤中的作用

Role of conventional protein kinase Cγ and its interacting protein 14-3-3γ in oxygen\|glucose deprivation-induced ischemic injury of primary cultured mouse cortical neurons

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