原代培养海马神经元周期素依赖性蛋白激酶5及其活性在低氧环境中的变化

Changes in the activity of cyclin-dependent kinase 5 in hippocampal neurons cultured primarily under hypoxia

目的:观察低氧对原代培养海马神经元存活力、周期素依赖性蛋白激酶5蛋白表达及其活性变化的影响。方法:实验于2005-01/12在解放军第二军医大学实验动物中心完成。取孕14～18dSD胎鼠的海马细胞进行原代培养,培养8～10d的细胞用于实验。取培养稳定阶段已分化成熟的大鼠海马神经元至低氧环境,继续培养12,24,36h,微孔酶标仪590nm处暗室内测定神经元存活力,免疫印迹测定周期素依赖性蛋白激酶5蛋白表达及其活性改变,每个时间点重复3次。结果:与常氧组比较,低氧组从继续培养12h起神经元存活力明显下降[(1.01±0.14),(0.77±0.15);(0.98±0.12),(0.68±0.11);(0.95±0.11),(0.61±0.08);P<0.01],周期素依赖性蛋白激酶5蛋白表达水平下降[(1.67±0.19),(0.82±0.08);(1.54±0.13),(0.75±0.06);(1.49±0.13),(0.70±0.11);P<0.01],活性明显增加[(1.76,3.56);(1.84,4.29);(1.95,6.04);P<0.01]。结论:低氧环境可导致神经元损伤,与周期素依赖性蛋白激酶5过度激活有关。

AIM：To observe the changes in neuronal survival ability, protein expression and activity of cyclin-dependent kinase 5 （cdk5） in hippocampus cultured primarily exposed to hypoxia environment. METHODS： The experiment was carried out in the Experimental Animal Center of Second Military University of Chinese PLA from January to December 2005. Neurons from the hippocampus of 14-18 day rat fetuses were cultured for 8-10 days, and then exposed to hypoxia environment for culture for 12, 24 and 36 hours, respectively. The survival ability of neurons was measured in a darkroom withmieropore reader at 590 nm; cdk5 protein level was estimated with Western blot methods and the activity of cdk5 was estimated for three times. RESULTS： Compared with the normal group, the survival ability of hippocampus neurons exposed to hypoxia environment was decreased significantly from 12 hours [（1.01±0.14）, （0.77±0.15）; （0.98±0.12）, （0.68±0.11）; （0.95±0.11）, （0.61±0.08）; P 〈 0.01], and the expression of cdk5 protein level in the hypoxia group was decreased significantly [（1.67±0.19）, （0.82±0.08）, （1.54±0.13）, （0.75±0.06）; （1.49±0.13）, （0.70±0.11）; P〈 0.01], while the activity of cdk5 was increased remarkably [（1.76, 3.56）; （1.84, 4.29）; （1.95, 6.04）; P 〈 0.01）. CONCLUSION： Hypoxia could cause hippocampus injury, which may be related with the increased level of cdk5 activity.