培养大鼠皮层神经元缺氧损伤与蛋白激酶活化关系的研究

目的检测神经元缺氧后膜性PKA(蛋白激酶A)和PKC(蛋白激酶C)活性变化,以及它们的特异性抑制剂对神经元凋亡率的影响.方法建立培养的神经元"缺血"模型,然后用不同浓度的特异性PKA抑制剂Rp-cAMP和特异性PKC抑制剂Calphostin C预孵育培养神经元再进行"缺血"实验,用放射酶分析法测定神经元缺氧后的膜性蛋白激酶活性,各组缺氧神经元的凋亡百分率由TUNEL荧光试剂盒测定.结果随着神经元缺氧时间的延长,神经元膜性PKA和PKC活性均增加;随着Calphostin C浓度的增加,细胞凋亡率显著增加;相反随着Rp-cAMP浓度的增加,细胞凋亡百分率显著减少.结论①PKA和PKC信号转导通路均参与了缺氧神经元损伤;②缺氧后培养神经元PKA和PKC对凋亡的调控功能相反,提示在培养的缺氧神经元中PKA和PKC信号转导属于单相控制系统,可能二者在细胞中的比例决定着缺氧神经元的存亡.

Roles of PKA and PKC in the process of anoxic apoptosis of cultured rat cortical neurons

Objective To detect the percentage of cultured neuron apoptosis after the neurons were treated with anoxia and specific inhibitors of protein kinase A (PKA) and protein kinase C (PKC). Methods After establishment of the model of neurons cultured under hypoxic condition, the neurons were cocultured with different concentrations of Rp-cAMP and Calphostin C, specific inhibitors of protein kinase A and C, respectively. Then neurons were cultured under an ischemic condition until the number of survived neurons, the activity of mPKA,and mPKC, and the apoptotic neurons stained by TUNEL in each group were observed. Results The activity of mPKA and mPKC significantly increased after the onset of hypoxia. With the increases in concentrations of Rp-cAMP or Calphostin C, the percentage of apoptotic neurons obviously decreased or increased, respectively. Conclusion The pathways of PKA and PKC signal transduction may participate in the hypoxic neuron injury. The functions of these two kinases are opposite for apoptotic regulation. It suggests that the signal transduction of PKA and PKC in hypoxic neurons belongs to a monophasic controlling system and the ratio of PKA to PKC in cells may determine the survival of hypoxic neurons.