不同底物对Sombati癫(癎)细胞模型整合素α2表达的影响

摘要

Objective To investigate the effect of different coating substrates on the integrin α2 expression level of hippocampal neurons in Sombati epileptic cell model.Methods After coated with laminin and poly-L-lysine as substrates on the tissue culture plates respectively, hippocampal neurons were isolated from 24 h Kunming mouse and cultured in vitro.The primary cultured neurons were identified by immunohistochemical technique, and cultured with Mg2+-free extracellular fluid for 3 h to prepare Sombati epileptic cell model.Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the integrin α2 mRNA expression of hippocampal neurons in each group on the eighth day.Results Before the model was prepared, neurons became full and large, processes connected closely, and neuronal network was formed at 5 d culture in vitro in laminin group, while the above appearances were seen at 7 d in poly-L-lysine group.Twenty-four hours after model preparation, neuronal migration phenomenon and "mesh-like" changes could be seen in all different groups.The ratio of the optical density of integrin α2 to β-actin could represent the relative expression level of integrin α2 mRNA.The ratio value in poly-L-lysine group, laminin group and poly-L-lysine + laminin group was 0.25 ± 0.03, 0.37 ± 0.05 and 0.48 ± 0.09, respectively.The expression level of integrin α2 mRNA in laminin group and poly-L-lysine + laminin group was all higher than that in poly-L-lysine group significantly (P = 0.005, 0.000; respectively).Conclusion In comparison of poly - L - lysine and laminin can strongly promote the formation of the neuronal network.Additional exogenous laminin can up - regulate integrin α2 mRNA expression of hippocampal neurons in Sombati epileptic cell model.%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α2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3h后制备Sombati癫(癎)细胞模型,逆转录-聚合酶链反应检测小同处理组海马神经元整合素α2 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24h,不同处理组神经元均町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α2 mRNA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、0.37±0.05和0.48±0.09,后两组整合素α2 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町诱导Sombati癫(癎)细胞模型海马神经元整合素α2mRNA表达上调.