CRMP-5-pEGFP-C2真核表达载体的构建及促神经元突起生长作用

发布时间：2018-07-27 10:15

【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(Collapsinresponse mediator protein-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6d SD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。
[Abstract]:Aim: to construct CRMP-5-pEGFP-C2 eukaryotic expression vector and to investigate the effect of collapse response regulatory protein -5 (Collapsinresponse mediator protein-5 (CRMP-5) overexpression on neuronal growth in rat hippocampal neurons. Materials and methods: the target gene cDNA was amplified by RT-PCR and cloned into pEGFP-C2 eukaryotic expression vector. Primary cultured rat hippocampal neurons were transiently transfected with CRMP-5-pEGFP-C2 eukaryotic expression vector by liposome method. Results: (1) CRMP-5 gene was amplified successfully, and the eukaryotic expression vector of CRMP-5-pEGFP-C2 was constructed. The sequence homology of CRMP-5-pEGFP-C2 and genebank was 100%. (2) after transient transfection with liposome, a small number of cells in the empty vector transfection group showed green fluorescence. There was no significant difference in length and number of branches between CRMP-5-pEGFP-C2 transfection group and blank vector group. The number of cells expressing green fluorescent protein in CRMP-5-pEGFP-C2 transfection group was higher than that in empty vector group. The neurites were longer than the other two groups, and most of the positive cells had processes more than ten times the diameter of the cell body, which interlaced with each other to form a neural network and extend to the distal end. Conclusion: (1) the eukaryotic expression vector of CRMP-5-pEGFP-C2 was successfully constructed. (2) CRMP-5 was expressed in the cell bodies and processes of hippocampal neurons. (3) when CRMP-5 was overexpressed, the number of secondary processes was increased. The results suggest that CRMP-5 may promote neuronal process growth and branches.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

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