目的:探讨Cdk5及p35在神经生长因子(NGF)撤退诱导的PC12细胞凋亡中的作用机制.方法:建立NGF撤退诱导的已分化PC12细胞凋亡模型,Western blotting检测Cdk5及p35在凋亡过程中表达变化情况,利用Cdk5特异性抑制剂Roscovitine预处理已分化PC12细胞,检测其对NGF撤退诱导的凋亡作用影响,向已分化PC12细胞转染真核表达质粒pCMV-p35-IRES-Cdk5,检测过表达CdkS/p35对PC12细胞凋亡的影响.结果:NGF撤退36h会引起已分化PC12细胞出现典型的DNA Ladder凋亡特征,MTT检测结果也显示,NGF撤退对PC12细胞的损伤呈时间依赖性;Roscovitine预处理已分化PC12细胞可以抑制NGF撤退诱导的细胞凋亡率,但不影响Cdk5/p35蛋白表达水平;向已分化PC12细胞中转染真核表达质粒后,能检测到Cdk5/p35蛋白的过表达,并引起PC12细胞出现凋亡样改变.结论:Cdk5及p35的活化与NGF撤退诱导的已分化PC12细胞凋亡过程密切相关,抑制Cdk5的活化有抑制细胞凋亡保护神经元的作用.%Aim;To investigate the roles of Cdk5/p35 in apoptosis of induced by NGF withdrawal. Methods: The models of differentiated PC12 cells apoptosis were established with NGF withdrawal and Western blotting were carried out to detect the expressions of Cdk5 and p35 during the apoptotic process. Roscovitine is the specific inhibitor of Cdk5, which was used for pretreatment of differentiated PC12 cells to determine the impact on apoptosis. The Eukaryotic expression plasmids pCMV-p35-IRES-Cdk5 were transfected into differentiated PC 12 cells and the effects on apoptosis of PC 12 cells were observed. Results : The typical feature of apoptosis, DNA Ladder could be detected at 36 h after NGF withdrawal. The results of MTT indicated that the damages to PC12 cells showed a time-dependent. After preincubation with Roscovitine, the rates of apoptosis were decreased, but the protein expression levels of Cdk5 and p35 did not change significantly. The overexpression of Cdk5/ p35 leaded to the occurrence of apoptosis in PC 12 cells that were transfcted with pCMV-p35-IRES-Cdk5 plasmids. Conclusion-.There is a close relationship between Cdk5/ p35 activation and apoptosis of differentiated PC12 cell induced by NGF withdrawal. Inhibition of activation of Cdk5 may contribute to reducing the rate of apoptosis and protecting neurons.
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