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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 investigate the role of classical cKKγ and its interacting protein 14-3-3γ in ischemic injury induced by oxygen-glucose deprivation (OGD) in primary cultured mouse cortical neurons. Methods cPKCγ knockout (cPKCγ - / -) mice and primary cultured cortical neurons were treated with 1hOGD / 24h hypoglycemic reoxygenation (R) to simulate the cell model of ischemia in vitro and the interference of 14-3-3γ inhibitor R18 The interaction between cPKCγ and 14-3-3γ and its effect on primary culture were verified by Co-IP, Western blotting and MTT assay. Effect of 1hOGD / 24hR Ischemia Injury in Rat Cerebral Cortical Neurons. Results Co-IP showed that 14-3-3γ interacted with cPKCγ in mouse cerebral cortex. 1hOGD / 24hR treatment induced cPKCγ protein in cPKCγ + / + mouse cortical neurons (P <0.05, n = 6 in each group). For cPKCγ - / - mice primary cultured cortical neurons, although the level of 14-3-3γ was significantly increased (P <0.05, n = 6 in each group), suggesting that cPKCγ may affect the level of 14- 3-3γ expression levels. In addition, the results of MTT assay showed that R18 (100μmol / L), a 14-3-3γ inhibitor, significantly increased the ischemic injury of cPKCγ - / - and cPKCγ + / + mice cortical neurons cultured in primary culture for 1hOGD / 24h <0.05, n = 6 for each group). Conclusions cPKCγ interacts with 14-3-3γ in cortical neurons of mice and affects the expression of 14-3-3γ protein. The expression of cPKCγ and 14-3-3γ in primary cultured cortical neurons of ischemic / Hypoxia plays a protective role.