皮质酮对原代培养的海马神经元及其Ca~(2+)/CaMKII的影响

在培养液中加入 10 7、10 6和 10 5浓度(mol/L)的皮质酮,采用MTT染色测定、Fura 2 /AM的荧光标记以及Western印迹的方法,观察了不同浓度的皮质酮作用下海马神经元形态学和细胞存活率的变化以及胞浆内游离钙离子浓度 Ca2+ ]i和CaMKII表达的变化规律, 探讨其对原代培养的大鼠海马神经元及其Ca2+ /CaMKII的影响和可能的机制。结果发现: 10 6、10 5浓度的皮质酮对海马神经元的形态学影响较大,与对照组比较,细胞存活率明显降低; Ca2+ ]i分别为 113. 1022±16. 9716、155.3794±20. 7727;CaMKII的表达也明显减少;三者的变化均显著 (P<0. 01 ),而 10 7浓度的皮质酮对上述指标影响不大 (P>0.05)。此外,相关性分析表明: Ca2+ ]i和CaMKII的表达呈现负相关(P<0. 05)。以上结果提示,皮质酮对大鼠海马神经元的作用存在浓度依赖效应,浓度越高,对大鼠海马神经元的损伤越大,同时也验证了皮质酮是啮齿类动物的主要的应激激素。

EFFECT OF CORTICOSTERONE ON PRIMARY CULTURED HIPPOCAMPAL NEURONS AND THEIR Ca ~(2+)/CaMKII EXPRESSION

After 10 -7, 10 -6or 10 -5mol/L of corticosterone (CORT) was added to culture medium, MTT assay, fluorescent labeling of Fura-2/AM and Western blotting were used to observe the change of cultured hippocampal neurons in morphology, the rate of survival, the cytosolic free calcium (Ca 2+]_i ) and the expression of CaMKII. The results showed the evident effect of 10 -6and 10 -5 mol/L of CORT on the morphology of hippocampal neurons. The survival rates of cells with both treatments markedly decreased. Ca 2+]_i is 113.1022±16.9716 or 155.3794±20.7727 for these two conditions and the expression of CaMKII was also reduced significantly(P<0.01). However no significant changes(P>0.05) in these paramcters were observed with 10 -7 mol/L CORT treatment. The correlation analysis shows that negative correlation exist between Ca 2+]_i and the expression of CaMKII(P<0.05). The results indicate that the effect of CORT on the hippocampal neurons is concentration-dependent, that is, the higher concentration of CORT, the more impairment it can induce in hippocampal neurons. At the same time, these data also support that CORT is main stressed hormone for the rodent.