CRMP-1真核表达载体的构建及其突起生长抑制作用

The Construction of CRMP-1 Eukaryotic Expression Vector and Growth Inhibiting Function on Neurite

为研究坍塌反应调节蛋白-1(collapsin response mediator protein-1,CRMP-1)对神经元突起生长的作用,构建了CRMP-1真核表达载体,以神经生长因子(NGF)诱导的PC12细胞为模型,采用基因转染、突起生长时差成像、突起提取和免疫印迹技术进行观察.结果显示,NGF诱导的PC12细胞具有典型的神经元形态特征,脂质体转染技术可成功地把CRMP-1基因导入细胞.过表达CRMP-1可明显抑制突起生长,促进突起坍塌,首先是细小突起缩短,然后是长突起,细胞突起的长度随CRMP-1蛋白表达时间延长呈逐渐缩短的趋势.突起提取液的测定显示,CRMP-1过表达的细胞较NGF诱导的和转染空载体的细胞突起明显减少(P<0.01).说明CRMP-1具有明显的突起生长抑制作用.

To investigate function of collapsin response mediator protein-1 （CRMP-1） on neurite outgrowth, the CRMP-I-phrGFP I/-N eukaryotic expression vector was constructed and transfected into PC12 cells induced by nerve growth factor. The lapse-time imaging and neurite extraction, as well as immunoblotting were utilized. The results demonstrated that PC12 cells were induced to be typical neurons by nerve growth factor. Lipofectamine could effectually transfect CRMP-1 into PC12 cells induced by nerve growth factor. Overexpression of CRMP-1 inhibited neurite outgrowth and collapsined the tiny neurite, then the longer neurite. Length of neurites demonstrated a tendency to be gradually shorten accompanying expression of CRMP-1 protein. Neurite extraction showed that cells overexpressing CRMP-1 possesed more and longer neurite（P 〈 0.01）, compared with cells only induced by nerve growth factor and transfected by expression vector without CRMP-1. These data suggested that CRMP-1 play an important role in inhibiting neurite outgrowth.