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

作　　者：章欣欣[1] 杨璇[1] 马维[1] 陈璐勔 韩松[1] 李俊发[1] 

机构地区：[1]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

出　　处：《解剖学报》2015年第2期145-150,共6页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(31171147;31471142);北京市自然科学基金资助项目(7141001;7144188);IATN国际合作种子基金资助项目(PXM2014-014226-000006);2013年北京市新世纪百千万人才工程培养经费B类资助项目(D08-016)

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1hOGD/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(Westernblotting)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1hOGD/24hR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;1hOGD/24hR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1hOGD/24hR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1hOGD/24hR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。Objective To explore the role of conventional protein kinase Cγ（ c PKCγ） and its possible interacting protein 14-3-3γ in 1hour oxygen-glucose deprivation（ OGD） /24 hours reoxygenation and glucose restoration（ R）-induced ischemic injury in primary cultured cortical neurons of mice. Methods Using c PKCγ gene knockout（ c PKCγ^-/- mice,1hour OGD /24 hours R-induced ischemic injury model of primary cultured cortical neurons,and the techniques of coimmunoprecipitation（ Co-IP）,Western blotting and 3-（ 4,5-dimethyl thiazole-2）-2,5-diphenyl four azole nitrogen bromine salt（ MTT） assay,the interaction of c PKCγ and 14-3-3γ and their effects on ischemic injury were determined. Results The Co-IP results confirmed the existence of c PKCγ interaction with 14-3-3γ in the cerebral cortex of mice; the decreased protein expression of c PKCγ was accompanied with the increase of 14-3-3γ protein levels in primary cultured cortical neurons from c PKCγ^＋/＋ice after 1hour OGD /24 hours R treatment（ P〈0. 05,n = 6 per group）. Although the 14-3-3γprotein level in cortical neurons from c PKCγ^-/-ice was higher than that from c PKCγ^＋/＋ice,1hour OGD /24 hours Rtreatment caused a significant decrease of 14-3-3γ protein level in cortical neurons from c PKCγ^-/-ice（ P〈0. 05,n = 6per group）,suggesting c PKCγ 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 /24 hours R-induced ischemic injury of primary cultured cortical neurons from both c PKCγ^＋/＋nd c PKCγ^-/-ice（ P〈0. 05,n = 6 per group）.Conclusion c PKCγ may interact with 14-3-3γ and affect 14-3-3γ protein expression level in cortical neurons of mice.c PKCγ and 14-3-3γ may protect primary cultured cortical neurons against 1hour OGD /24 hours R-induced ischemic injury.

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 

分 类 号：R338[医药卫生—人体生理学]