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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。
Cortisone concentrations of 10 7, 10 6 and 10 5 (mol / L) were added to the culture medium. MTT staining, fluorescent labeling of Fura 2 / AM and Western blotting were used to observe the effects of different concentrations of corticosterone The changes of morphology and cell viability of hippocampal neurons and the changes of intracellular free calcium concentration [Ca2 +] i and CaMKII expression were investigated to investigate the effect of Ca2 + / CaMKII on primary cultured hippocampal neurons Possible mechanism. The results showed that 10 6,10 5 corticosterone had a significant effect on the morphological changes of hippocampal neurons. Compared with the control group, the cell viability decreased significantly. [Ca2 +] i was 113.102 ± 16.9716 and 155.3794 ± 20. 7727; CaMKII expression was also significantly reduced; changes in the three were significant (P <0.01), while 10 7 corticosterone had little effect on the above indicators (P> 0.05). In addition, correlation analysis showed that there was a negative correlation between [Ca2 +] i and CaMKII (P <0.05). The above results suggest that corticosterone has a concentration-dependent effect on the hippocampal neurons in rats. The higher the concentration, the greater the damage to the hippocampal neurons in rats, and the corticosterone is also found to be the major stress hormone of rodents .