基因水平下调PP2A水平可显著抑制海马神经元轴突生长

Downregulation of PP2A level by gene knockout could inhibit hippocampal neuronal axon outgrowth significantly

目的探讨基因水平下调PP2A对胎鼠海马神经元轴突生成的影响。方法选取体外培养原代海马神经元为研究模型,首先构建PP2A催化亚基特异性干扰RNA及其对照(si-PP2A/ssi-PP2A)质粒,选择在种植前下调PP2A水平,观察其对海马神经元轴突生成是否有作用;原代细胞采用Amaxa大鼠神经元核电转试剂盒分别转染EGFP-ssi PP2Ac和EGFP-si PP2Ac,神经元种植培养48 h后固定做免疫荧光双标,分别标记轴突特异性标记物Tau-1和树突特异性标记物MAP-2,观察基因下调PP2A水平对神经元轴突生成的影响。结果原代海马神经元转染48 h后对照转染组海马神经元神经元轴突和树突均已形成,而干扰RNA转染组神经元轴突生长受到显著抑制,而树突生长未受到明显影响;si-PP2A转染组(干扰转染组)神经元轴突长度仅为对照转染组的38%,单个神经元的平均轴突数目也从正常1.0/neuron下降到0.5/neuron。结论基因下调PP2A水平显著抑制了原代海马神经元轴突生成,结合前期药物下调研究结果提示维持细胞内正常PP2A水平在海马神经元轴突生成中起重要作用。

Objective To explore the effect of downregulation of PP2A level by gene konckout on fetal rat hippocampal neuronal axon outgrowth.Methods The primary hippocampal neuron systems was chose as the model for study.First the PP2A catalytic subunit specific RNA interference plasmid and its control （si-PP2A/ssi-PP2A） were built,and then the effect of siPP2A on the neuron axon formation was observed when the plasmids were transfected before cell plating.Primary hippocampal neurons were transfected with si-PP2A or ssi-PP2A by Amax rat neuron nuclear transfer kit,and the neurons were fixed by immunofluorescence after 48 h.Then the cells were labeled with axon specific marker Tau-1 and dendritic specific marker MAP-2.The effect of PP2A level on the axon formation was observed.Results Hippocampal neurons were transfected with si-PP2A or ssi-PP2A for 48h,then cells were measured the alterations of axons by using double immunofluorescence.Data showed that Axons were formed after culturing for 48h in ssi-PP2A transfected group under the present conditions.However,the formation of axons were inhibited significantly when transfected with siPP2A,and there was no effect on the dendrites formation.Further statistical analysis data showed that siPP2A-transfected axon average length were only 38% of control group （ssiPP2A）,and the average number of axons of single neuron also dropped from the normal 1.0/neuron to 0.5/neuron.Conclusion Gene downregulation of PP2A significantly inhibited axon formation in primary cultured hippocampal neurons,combined of preliminary study results,thus results indicated that maintaining the normal level of PP2A in the cells played an important role in the axon formation of hippocampal neurons.