血液溶解产物对原代培养的小鼠脑皮层神经元的影响

Effects of hemolysate on primary cultured mouse cortical neurons

目的 探讨血液溶解产物对原代培养的小鼠脑皮层神经元的影响及其机制。方法 分离培养新生1～3 d C57BL/6小鼠脑皮层神经元并进行鉴定;加入不同浓度的血液溶解产物,通过细胞形态学观察、细胞计数法评估细胞生长情况;Hochest33342染色法及流式细胞术检测细胞凋亡;Western blot法检测凋亡通路相关蛋白cleaved caspase-3的表达情况。结果原代培养的小鼠脑皮层神经元Neu N阳性细胞率为（92.24±1.16）%;形态学显示随着血液溶解产物的浓度增加细胞损伤程度增大,贴壁细胞数减少,且Hochest33342染色阳性率增加,细胞凋亡率增加,凋亡通路相关蛋白cleaved caspase-3的表达也增加,且呈浓度依赖性,浓度越高,凋亡蛋白表达越高。结论 血液溶解产物能抑制脑皮层神经元的生长,其机制可能是通过上调凋亡通路相关蛋白的表达而诱导细胞凋亡来实现的。

Objective To investigate the effect of hemolysate on primary cultured mouse cortical neurons. Methods The cortical neurons were isolated from 1N3 days newborn C57BL/6 mice, cultured and identified. The cultured cortical neurons were treated with different concentrations of hemolysate. Cell survival status was assessed by morphological observation and cell count methods. Cell apoptosis was measured by Hochest33342 fluorescent stain and flow cytometry. The expression of apoptotic pathway related protein, cleaved caspase-3, were detected by Western blot. Results The rates of NeuN-positive neurons were （92.24± 1.16）% 7 days after the culture of mouse cortical neurons. Hemolysate significantly increased the damage to the neurons and reduced the number of adherent neurons in a concentration dependent manner. Hemolysate significantly increased the rates of Hochest33342-positive neurons and the apoptotic rates of primary cultured mouse cortical neurons, and up-regulated the expression of apoptotic pathway related protein, cleaved caspase-3, in a concentration dependent manner. Conclusions That Hemolysate can significantly inhibit the survival of cortical neurons may be via up-regulated the expression of apoptotic pathway related protein, cleaved caspase-3.