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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
章欣欣 杨璇 马维 陈璐勔 韩松 李俊发*
解剖学报 ›› 2015, Vol. 46 ›› Issue (2) : 145-150.
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
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。
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.
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
Primary cultured cortical neuron / Oxygen-glucose deprivation / Ischemic injury / Conventional protein kinase Cγ / 14-3-3γ / Western blotting / Mouse
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