脑衰反应调节蛋白-2(CRMP-2)促进海马神经元轴突生长

摘要

Objective To study the effect of collapsin response mediator protein ( CRMP2) on primary hippocampal neurons,and to further explore the role of CRMP-2 in the outgrowth of hippocampal neurons.Methods We chose primary hippocampal neuron systems as the model for study. Hippocampi from 18-day embryonic rats were collected and digested with trypsin for hippocampal neurons. We transfected in the hippocampal neurons with EGFP or wtCRMP2, or Thr514-phosphomimic CRMP2 (T514D-CRMP2) by nuclear transfection, and then measured the alterations of axons by using immunofluorescence. The neurons were then stained with EGFP and Tau-1 at 72h and measured the morphology with Laser confocal microscope. ResultsThe neurons transfected with E(JFP growed normally, however, the axon length, numbers and the percent ofmultiple axons were all increased by transfecting with wtCRMP2. But the increase was not detected when T514D-CRMP2 was transfected. Conclusions Overexpressing wtCRMP-2 could promote axon outgrowth, but phosphomimic CRMP2 couldn' t, indicating that non-phosphorylating CRMP2 promotes axon outgrowth.%目的 研究脑衰反应调节蛋白2(collapsin response mediator protein,CRMP-2)对原代海马神经元轴突生长的影响,探讨CRMP-2在神经元轴突生成中的作用.方法 每次原代取孕18 d SD大鼠1只,每只孕鼠含胎鼠约12～15只.显微镜下分离12～15只胎鼠双侧海马组织,消化法培养原代海马神经元.原代海马神经元培养采用核电转实验转染绿色荧光蛋白(enhanced green fluorescence protein,EGFP)和野生型CRMP-2(wild type (wt)CRMP2)和突变型T514D-CRMP2 (突变体,模拟失活型CRMP-2).培养72h固定做免疫荧光双标,分别绿色荧光蛋白(EGFP)和轴突标志物Tau-1,采用激光共聚焦显微镜和普通光学显微镜观察海马神经元形态变化.结果 转染EGFP载体的对照组神经元正常发育,培养至72 h,神经元轴突特异性表达标志物蛋白Tau-1;过表达了wtCRMP-2的神经元除了生成一条长的特异性表达Tau-1的轴突,另外一条突起也发育成了特异性表达Tau-1的轴突;而过表达了失活型CRMP-2的神经元发育与正常组相比无差异.结论 野生型CRMP-2可明显促进轴突生长,而转染模拟磷酸化CRMP-2的突变体T514D-CRMP2 则没有显示任何的促进作用.