CRMP-5-pEGFP-C2真核表达载体的构建及促神经元突起生长作用.pdfVIP

中文摘要 目的： 构建 CRMP-5-pEGFP-C2 真核表达载体，探讨坍塌反应调节蛋白-5(Collapsin responsemediatorprotein-5,CRMP-5)过表达情况下对大鼠海马神经元突起生长的影响。 材料和方法： 提取新生6dSD大鼠脑海马组织总RNA,RT-PCR扩增目的基因cDNA并克隆到 pEGFP-C2 真核表达载体上。原代培养大鼠海马神经元，脂质体法瞬时转染CRMP-5- pEGFP-C2真核表达载体，通过绿色荧光蛋白示踪观察其表达部位及突起生长情况。 结果： (1) 成功扩增CRMP-5基因cDNA，构建了CRMP-5-pEGFP-C2真核表达载体，经 测序与genebank的序列同源性为100%。(2) 脂质体法瞬时转染后，空载体转染组少量 细胞显示绿色荧光，且均匀分布于胞体和突起，突起长度和分支数目与空白对照组无明 显差异；CRMP-5-pEGFP-C2转染组表达绿色荧光蛋白的细胞数量较空载体组多，荧光 于胞体、突起末端及分支处较突起中段强，细胞突起明显较其他两组绵长，多数阳性细 胞具有超过胞体直径数十倍的突起，相互交错形成神经网络，并向远端延伸。 结论： (1) 成功构建了CRMP-5-pEGFP-C2真核表达载体。(2)CRMP-5 表达于海马神经元 的胞体和突起。(3) CRMP-5 过表达时细胞突起延伸明显且次级突起数目增多，表明 CRMP-5可能具有促进神经元突起生长和分支的作用。 关键词： 坍塌反应调节蛋白-5， 真核表达载体， 突起， 生长， 细胞骨架， 神经元，海 马，大鼠 I ABSTRACT ABSTRACT AABBSSTTRRAACCTT Objective: Objective: OObbjjeeccttiivvee:: To construct the CRMP-5-pEGFP-C2 eukaryotic expression vector and investigate the positive effect of up-regulation of the CRMP-5 gene on neurite outgrowth of hippocampus neurons inrats. Materials andmethods: Materials andmethods: MMaatteerriiaallssaannddmmeetthhooddss:: To extract the total RNA from the hippocampus tissue of a 6 days SD rats and amplify the cDNA of CRMP-5 gene by RT-PCR technique, the products were cloned into pEGFP-C2 eukaryotic expression vector. The recombined vector was then transiently transfected into hipppcamups neurons, at last, to observe the localization of CRMP-5 protein and changes in neuritebyfluorescencemicroscope. Results: Results: RReessuullttss:: （1）The cDNA of CRMP-5 gene were amplified successfully, and the CRMP-5- pEGFP-C2 eukaryotic expression vector was constructed, the basal pairs of which were absolutely homologous with the sequence acquired from genebank. (2) After transfection, the fewer cells transfected by pEGFP-C2 v