Calphostin C对培养神经元缺氧后Bcl-2表达及神经元凋亡的研究

Influence of protein kinase C on the expression of Bcl - 2 and anoxic cell apoptosis of cultured rat cortical neuron

目的：探讨蛋白激酶Ｃ（ＰＫＣ）对神经元缺氧凋亡的影响及作用机制。方法：建立体外培养孕Ｗｉｓｔａｒ大鼠皮层神经元模型及培养神经元缺氧模型，在此基础上观察神经元存活率、Ｂｃｌ—２和凋亡ＤＮＡ表达的规律；然后用３种不同浓度的ＰＫＣ催化亚基特异性抑制剂 Ｃａｌｐｈｏｓｔｉｎ Ｃ预孵育培养神经元后进行缺血处理，观察神经元上述指标的改变。结果：随着缺氧时间的延长和 Ｃａｌｐｈｏｓｔｉｎ Ｃ浓度的增加，培养神经元的存活数显著下降、Ｂｃｌ—２免疫组化阳性细胞数明显减少、ＴＵＮＥＬ荧光染色平均荧光强度显著升高。结论：（１）ＰＫＣ和Ｂｃｌ－２均参与了缺氧神经元凋亡；（２）ＰＫＣ抑制剂Ｃａｌｐｈｏｓｔｉｎ Ｃ可加重缺氧神经元凋亡；（３）ＰＫＣ的激活在缺氧神经元的凋亡中起保护作用，且该作用可能是通过促进Ｂｃｌ—２表达实现的。

Objective: To investigate the mechanism that how protein kinase C(PKC) affects the anoxic neuron apoptosis. Methods: First, in vitro models of neuronal culture and anoxic neuronal culture were established. Second, three different concentrations of Calphostin C were used to cocultured with the neurons. Finaly, the number of survival neurons and the expression of Bcl - 2 and apoptotic DNA fragments were detected in each group. Results: With hypoxic time prologation and the concentration of Calphostin C increasing, the number of survival neurons decreased obviously, the number of neurons that positively stained by Bcl - 2 decreased. And on the contrary the neurons' average optical density of TUNEL was significantly increased. Conclusions: (1) Both PKC and Bcl - 2 are participate in the process of anoxic neuron apoptosis. (2) Calphostin C can aggravate anoxic neuron apoptosis. (3) The activation of PKC can protect anoxic neuron apoptosis through promoting the expression of Bcl - 2.