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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18d 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72h,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。
Objective To investigate the effect of CRMP-2 on axon growth of primary cultured hippocampal neurons and to explore the role of CRMP-2 in neuronal axon production. Methods Each primary pregnancy 18 SD rats, each with about 15 fetuses. Under the microscope, 12 ~ 15 fetal rat bilateral hippocampus tissues were isolated and primary cultured hippocampal neurons were cultured by digestion. Primary cultured hippocampal neurons were transfected with enhanced green fluorescence protein (EGFP) and wild-type CRMP-2 (wild type (wt) CRMP2) and mutant T514D-CRMP2 Type CRMP-2). Cultured 72h immobilized double immunofluorescence labeling, respectively, green fluorescent protein (EGFP) and axon marker Tau-1, using laser scanning confocal microscope and optical microscopy to observe the morphological changes of hippocampal neurons. Results The neurons of the control group transfected with EGFP vector developed normally. After cultured for 72 hours, the neuronal axon specifically expressed Tau-1, and the neurons overexpressing wtCRMP-2 not only produced a long and specific Tau- 1 axons. The other protrusion also developed axons that specifically expressed Tau-1. The neurons overexpressing inactivated CRMP-2 showed no difference compared with that of normal control. Conclusions Wild-type CRMP-2 can significantly promote axon growth, whereas mutant T514D-CRMP2 transfected with mock phosphorylated CRMP-2 did not show any promotion.