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目的探讨不同底物对经体外培养的Sombati癫癎细胞模型海马神经元整合素α_2表达水平的影响及意义。方法以层黏连蛋白和多聚L-赖氨酸作为底物于体外培养新生小鼠海马神经元,至第7天时行神经元纯度鉴定;无镁细胞外液继续培养3 h后制备Sombati癫癎细胞模型,逆转录-聚合酶链反应检测不同处理组海马神经元整合素α_2 mRNA相对表达变化。结果模型制备前,经体外培养至第5天的层黏连蛋白组神经元胞体增大、饱满,突起连接紧密,神经网络形成,多聚L-赖氨酸组则体外培养至第7天时方出现上述表现;模型制备后24 h,不同处理组神经元均可见迁移现象和神经网络“网格”样改变,但不同处理组神经元形态无明显差异。以整合素α2与β-肌动蛋白光密度值之比值代表整合素α_2mRNA相对表达量,多聚L-赖氨酸组、层黏连蛋白组和多聚L-赖氨酸+层黏连蛋白组分别为0.25±0.03、037±0.05和0.48±0.09,后两组整合素α_2 mRNA表达水平高于多聚L-赖氨酸组,且差异有统计学意义(p=0.005,0.000)。结论与多聚L-赖氨酸组相比,层黏连蛋白对经体外培养的原代神经元贴壁及轴突连接具有较强的促进作用。添加外源性层黏连蛋白可诱导Sombati癫癎细胞模型海马神经元整合素α_2mRNA表达上调。
Objective To investigate the effect of different substrates on the expression of integrin α_2 in hippocampal neurons cultured in vitro in Sombati epileptic cell model. Methods The hippocampal neurons of neonatal mice were cultured in vitro with laminin and poly-L-lysine as substrates. The purity of neurons was identified by day 7, and the cells were cultured for 3 hours without magnesium extracellular solution. Sombatide癎 cell model and reverse transcription - polymerase chain reaction (RT - PCR) to detect the relative expression of integrin α_2 mRNA in hippocampal neurons of different treatment groups. Results Before the model was prepared, the neuronal soma of the laminin group cultured in vitro until day 5 was enlarged, full, the process of neurite formation was close, the neuronal network was formed, and the poly-L-lysine group was cultured in vitro until day 7 The results showed that at 24 h after model preparation, the migration of neurons in different treatment groups and the change of neural network “grid ” were observed. However, there was no significant difference in neuronal morphology among different treatment groups. The ratio of integrin alpha 2 to beta-actin OD was used to represent the relative expression of integrin alpha 2 mRNA, poly-L-lysine group, laminin group and poly-L-lysine plus laminin Group were 0.25 ± 0.03,037 ± 0.05 and 0.48 ± 0.09 respectively. The expression of integrinα_2 mRNA in the latter two groups was higher than that in poly-L-lysine group (p = 0.005,0.000). Conclusions Laminin has a strong promoting effect on adherent and axon junctions of primary neurons cultured in vitro compared with poly-L-lysine group. Addition of exogenous laminin can up - regulate the expression of integrin α_2 mRNA in the hippocampal neurons of Sombati epileptic septal cells.