体外原代培养大鼠胚胎脊髓神经细胞机械损伤后的凋亡与caspase-3的表达

为了研究体外培养大鼠胚胎脊髓神经细胞损伤前后的凋亡变化及caspase-3的表达情况,本实验建立了一个模拟脊髓横断损伤后脊髓组分-原代培养的脊髓神经细胞机械损伤模型,并用Hoechst33342/PI双染法检测脊髓神经细胞的凋亡变化,用免疫荧光染色方法检测caspase-3的表达。结果显示:(1)损伤前几乎未见凋亡的神经细胞,损伤后6 h凋亡细胞开始出现,损伤后12 h明显增多,1 d时达高峰,但损伤后3 d凋亡细胞开始呈明显下降的趋势,至损伤后7 d又开始增加,一直持续到损伤后14d;(2)相应时刻caspase-3表达趋势的改变与脊髓神经细胞凋亡趋势的变化基本相同;(3)经过caspase-3抑制剂Ac-DEVD-CHO干预后,凋亡细胞的数量与caspase-3的表达均减少,并呈剂量依赖性。以上结果提示,机械损伤可以诱发体外培养的脊髓神经细胞凋亡,且此凋亡的发生可能与caspase-3的表达有关。

Apoptosis and caspase-3 expression of primary cultured spinal cord neurons of rat embryo following mechanical injury in vitro

To study the changes of apoptosis and caspase-3 expression of primary cultured spinal cord neurons of rat embryo following mechanical injury in vitro,a model of mechanical injury to spinal cord component-primarily cultured spinal cord neurons,mimicking the neuronal injury after transectional injury of the spinal cord,were established.And the apoptosis and caspase-3 expression of the neurons were investigated separately by Hoechst33342/PI double staining and immunofluorescence methods.The results showed that:(1) Few apoptotic neurons were found before the injury.Apoptotic cells began to appear 6 hours after the injury.The number of apoptotic cells increased significantly 12 hours after the injury,and reached the maximum on the first day after the injury.However,apoptotic cells showed a tendency to decrease significantly in number on the third day after the injury.On the 7th day after the injury,apoptotic cells began to increase again.And this increase continued till the 14th day after the injury;(2)The changes of the caspase-3 expression after the injury were almost consistent with those of the apoptosis of the spinal cord neurons accordingly on all time-points;(3)After the intervention of caspase-3 inhibitor,Ac-DEVD-CHO,the number of apoptotic cells and the caspase-3 expression were dose-dependently reduced.The above results indicate that apoptosis of cultured spinal cord neurons can be induced by mechanical injury,which may be associated with caspase-3 expression.