p85a基因对小鼠大脑皮层投射神经元迁移的影响

Research on p85a Gene in Radial Migration of Cortical Projection Neurons of the Mouse Cortex

Pi3k/Akt信号通路是近年来发现的参与细胞增殖、调控的重要通路,Pi3k激活可介导多种细胞功能,Pi3k由p85a和p110构成。该研究通过干扰p85a基因的表达,探讨其在小鼠大脑皮层投射神经元迁移中的作用。首先,构建p85a基因的对照质粒(scramble)、siRNA质粒(sip85a-1、sip85a-2)和过表达质粒(OEp85a);接着转染N2a细胞,48 h后,用定量PCR方法检测p85a基因mRNA的表达情况。随后,将sip85a-1、sip85a-2、OEp85a质粒分别转入小鼠大脑,4 d后,借助免疫荧光方法检测皮层神经元的迁移情况。定量PCR结果显示:与对照相比,转染sip85a-1、sip85a-2均能显著降低N2a细胞中p85a基因的mRNA表达,抑制效率约为40%(P<0.05);而转染OEp85a质粒后,能显著增加p85a基因mRNA的表达,约为对照组的12倍(P<0.01)。胚胎电转结果中,各区EGFP阳性神经元数目定量分析显示,sip85a-1、sip85a-2、OEp85a质粒均能显著抑制神经元的迁移(P<0.05)。大脑发育阶段中,p85a基因在适当范围内,对平衡神经元迁移过程中起着重要作用。

In recent years, Pi3k/Akt signaling pathway has been found vitally in cell multiplication and regulation. Activation of Pi3k can modulate a variety of cellular functions. Pi3k consists of p85a and p110. In this study, we aimed to determine whether p85a regulates the early migration of cortical projection neurons in mice by interruption the expression ofp85a. First, we designed a scrambled shRNA plasmid as a control, two short hairpin RNAs （sip85a-1, sip85a-2） construct againstp85a and an overexpression plasmid （OEp85a）; Constructs were tested by transiently transfecting N2a cells. We detected the p85a mRNA level by Real-time PCR analysis. Then, we used two effective shRNAs to determine whether p85a was involved in the migration of cortical neurons. Real-time PCR analysis showed that p85a mRNA level decreased significantly after transfection with the plasmids expressing sip85a-1 （40%） and sip85a-2 （40%） compared to control （P〈0.05）. Conversely, p85a mRNA level increased significantly after transfection with OEp85a （P〈0.01）. In order to quantify in utero electroporation results, we also counted EGFP-positive cells in different bins. The results showed that all of sip85a-1, sip85a-2 and OEp85a could impair radial migration significantly （P〈0.05）. These observations indicate that balance ofp85a has vital functions in regulating migration of cortical neurons during brain development.